3-substituted piperidine compounds for cbl-b inhibition, and use thereof

ABSTRACT

Compounds, compositions, and methods for use in inhibiting the E3 enzyme Cbl-b in the ubiquitin proteasome pathway are disclosed. The compounds, compositions, and methods can be used to modulate the immune system, to treat diseases amenable to immune system modulation, and for treatment of cells in vivo, in vitro, or ex vivo. Also disclosed are pharmaceutical compositions comprising a Cbl-b inhibitor and a cancer vaccine, as well as methods for treating cancer using a Cbl-b inhibitor and a cancer vaccine; and pharmaceutical compositions comprising a Cbl-b inhibitor and an oncolytic virus, as well as methods for treating cancer using a Cbl-b inhibitor and an oncolytic virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of U.S.provisional application numbers: 62/831,392, filed Apr. 9, 2019;62/866,909, filed Jun. 26 2019; 62/880,267, filed Jul. 30, 2019;62/888,845, filed Aug. 19, 2019; and 62/888,870, filed Aug. 19, 2019,wherein the contents of each are incorporated herein by reference intheir entirety.

TECHNICAL FIELD OF THE INVENTION

Provided herein are compounds and compositions for inhibition of theCbl-b enzyme and methods of use thereof in modulating the immune system,treatment of diseases, and treatment of cells in vivo, in vitro, or exvivo. Also provided herein are pharmaceutical compositions, kits, andmethods of treating cancer comprising a combination of an inhibitor ofthe Cbl-b enzyme and a cancer vaccine; and pharmaceutical compositions,kits, and methods of treating cancer comprising a combination of aninhibitor of the Cbl-b enzyme and an oncolytic virus.

BACKGROUND

The ubiquitin proteasome pathway is a complex system involved in theregulation of protein function and catabolism. Proteins in eukaryoticcells are conjugated with ubiquitin, a 76 amino acid, 8.5 kilodaltonprotein. This conjugation, known as ubiquitination, results in alteredfunction or degradation of the target protein. Ubiquitination of thetarget protein occurs via a coupled series of reactions involvingubiquitin and a set of enzymes known as E1, E2, and E3 enzymes.Ubiquitin is activated by the ubiquitin-activating enzyme, or E1 enzyme.Ubiquitin is then transferred to a ubiquitin-conjugating enzyme, or E2enzyme. Finally, a ubiquitin ligase, or E3 enzyme, promotes the transferof ubiquitin from the E2 enzyme to the target protein.Polyubiquitination of the target protein predominantly serves as asignal leading to degradation of the ubiquitin-conjugated protein by theproteasome, where it undergoes proteolysis. Ubiquitination by E3 ligasescan also result in altered protein activity, interactions, orlocalization. Ubiquitination regulates diverse biology including celldivision, DNA repair, and cellular signaling.

The synthesis and degradation of proteins in the cell is critical forcell cycle regulation, cell proliferation, apoptosis, and many othercellular processes. Thus, the ability to modulate the ubiquitinproteasome pathway offers a wealth of opportunities to intervene indisease processes. Mechanisms for intervention can include enhanceddegradation of oncogene products, reduced degradation oftumor-suppressor proteins, modulation of immune cell response, andmodulation of anti-tumor immune responses.

Therapeutic cancer vaccines have been evaluated in numerous clinicaltrials. However, only two therapeutic cancer vaccines have been licensedfor use in the United States. In particular, the Bacillus of Calmetteand Guerin strain of Mycobacterium bovis has been approved for treatmentof bladder cancer, and an ex vivo-activated, autologous cell vaccine hasbeen approved for treatment of prostate cancer. Even so, the responserates and overall survival of patients treated with cancer vaccines areconsiderably lower than desirable. Thus, what is needed in the art aremethods of improving the efficacy of cancer vaccines.

Although numerous clinical trials employing an oncolytic virus to treatcancer have been conducted, only one oncolytic virus has been licensedfor use in the United States and Europe. In particular, talimogenelaherparepvec is a genetically modified herpes simplex virus approvedfor treatment of melanoma. However, even talimogene laherparepvec hasnot been shown to improve overall survival or to benefit patients withvisceral metastases. Thus, what is needed in the art are methods ofimproving the efficacy of oncolytic virus therapy.

Approximately 35 E2 enzymes and over 500 E3 enzymes are encoded in thehuman genome. Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is anE3 ubiquitin ligase that negatively regulates T-cell activation (Wallneret al., Clin Dev Immunol, 2012: 692639). Discovery of agents thatmodulate E2 or E3 enzymes accordingly provides the potential fortherapies directed against disease processes involving a particular E2or E3 enzyme. The present patent application is directed to agents thatinhibit one such E3 enzyme, Casitas B-lineage lymphoma proto-oncogene-b(Cbl-b); agents that inhibit Cbl-b, for use in combination with cancervaccines, and to pharmaceutical compositions comprising Cbl-b inhibitorsand cancer vaccines; and agents that inhibit Cbl-b, for use incombination with oncolytic viruses, and to pharmaceutical compositionscomprising Cbl-b inhibitors and oncolytic viruses.

SUMMARY OF THE INVENTION

Disclosed herein are compounds and compositions for inhibition of theCbl-b enzyme and methods of use thereof in modulating the immune system,treatment of diseases, and treatment of cells in vivo, in vitro, or exvivo. Also disclosed herein are methods for use of a Cbl-b inhibitor intreating cancer. In brief, the Cbl-b inhibitor may be administered to anindividual with cancer, either alone or as part of a combinationtherapy, with one or more of an immune checkpoint inhibitor, ananti-neoplastic agent, and radiation therapy. Additionally, cellstreated in vivo and/or in vitro with a compound or composition asdisclosed herein may be used in adoptive cell therapy for treatingcancer.

Disclosed herein are compounds of Formula (I):

-   -   or a tautomer thereof, or a pharmaceutically acceptable salt        thereof, wherein:

is

-   -   X is CH or N;    -   Z¹ is CH or N;    -   Z² is CH or N;    -   R^(1a) and R^(1b) are independently H, C₁-C₆ alkyl, C₁-C₆        haloalkyl, or C₁-C₆ alkyl-OH;    -   R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkyl-OH,        C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl);    -   R^(2b) is H, halo, or C₁-C₆ alkyl;    -   or R^(2a) and R^(2b) are taken together with the carbon atom to        which they are attached to form a spiro 3- to 6-membered        heterocyclyl or a spiro C₃-C₆ cycloalkyl,        -   wherein at least one of the atoms of the spiro heterocyclyl            which is adjacent to the connecting piperidinyl ring is            carbon;    -   R^(3a) and R^(3b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form C₃-C₄ cycloalkyl;    -   R⁴ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH;

is

and Y is CR^(5a)R^(5b) or S;

-   -   or

is

and Y is a bond;

-   -   W is O or a bond;    -   R^(5a) and R^(5b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form a C₃-C₆ cycloalkyl;    -   R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁷ is H, halo, C₃-C₆ cycloalkyl, —NH-(3- to 6-membered        heterocyclyl), —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl), —O-(3-        to 6-membered heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆        cycloalkyl);    -   R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁹ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and    -   R¹⁰ is —CF₃ or cyclopropyl.

In some embodiments,

is

In some embodiments, Z¹ is CH. In some embodiments, Z¹ is N.

In some embodiments,

In some embodiments,

is

In some embodiments, Z² is CH. In some embodiments, Z² is N.

In some embodiments,

is

In some embodiments,

is

and Y is CR^(5a)R^(5b) or S. In some embodiments, W is a bond or O.

In some embodiments,

is

and Y is a bond.

In some embodiments, R⁸ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄cycloalkyl. In some embodiments, R⁸ is —CH₃, —CF₃, or cyclopropyl.

In some embodiments, X is CH. In some embodiments, X is N.

In some embodiments, Y is a bond. In some embodiments, Y is CR^(5a)R⁵.In some embodiments, Y is S.

In some embodiments, R^(5a) and R^(5b) are independently H, halo, orC₁-C₃ alkyl, or R^(5a) and R^(5b) are taken together with the carbonatom to which they are attached to form a C₃-C₄ cycloalkyl.

In some embodiments, R^(5a) and R^(5b) are independently H, F, or —CH₃;or R^(5a) and R^(5b) are taken together with the carbon atom to whichthey are attached to form cyclopropyl.

In some embodiments, R^(5a) and R^(5b) are independently H or F.

In some embodiments, R^(1a) and R^(1b) are independently H, C₁-C₃ alkyl,C₁-C₃ haloalkyl, or C₁-C₃ alkyl-OH. In some embodiments, R^(1a) andR^(1b) are independently H, —CH₃, —CF₃, or —CH₂OH. In some embodiments,R^(1b) is H.

In some embodiments, R^(2a) is —CN, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃alkyl-OH, C₁-C₃ alkyl-CN, or —(C₁-C₃ alkylene)-O—(C₁-C₃ alkyl). In someembodiments, R^(2a) is —CN, —CH₃, —CF₃, —CH₂OH, —CH₂CN, or —CH₂—O—CH₃.

In some embodiments, R^(2b) is H, halo, or C₁-C₃ alkyl. In someembodiments, R^(2b) is H, F, or —CH₃.

In some embodiments, R^(2a) and R^(2b) are taken together with thecarbon atom to which they are attached to form a spiro 4- to 5-memberedheterocyclyl or a spiro C₃-C₄ cycloalkyl. In some embodiments, R^(2a)and R^(2b) are taken together with the carbon atom to which they areattached to form spiro cyclopropyl,

In some embodiments, R^(3a) and R^(3b) are independently H, halo, orC₁-C₃ alkyl, or R^(3a) and R^(3b) are taken together with the carbonatom to which they are attached to form C₃-C₄ cycloalkyl. In someembodiments, R^(3a) and R^(3b) are independently H, F, or —CH₃; orR^(3a) and R^(3b) are taken together with the carbon atom to which theyare attached to form cyclopropyl.

In some embodiments, R⁴ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃alkyl-OH. In some embodiments, R⁴ is H, —CH₃, —CF₃, or —CH₂OH.

In some embodiments, R⁶ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄cycloalkyl. In some embodiments, R⁶ is —CH₃, —CHF₂, or cyclopropyl.

In some embodiments, R⁷ is H, halo, C₃-C₄ cycloalkyl, —NH(4- to5-membered heterocyclyl), —NH(C₁-C₃ alkyl), —NH(C₃-C₅ cycloalkyl),—O(C₁-C₃ alkyl), —O(4- to 5-membered heterocyclyl), or —O(C₃-C₅cycloalkyl). In some embodiments, R⁷ is H, Cl, cyclopropyl, —NH(CH₂CH₃),—NH(cyclopropyl), —OCH₂CH₃, —O(cyclopropyl),

In some embodiments, R⁹ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃alkyl-OH. In some embodiments, R⁹ is H, —CH₃, —CF₃, or —CH₂OH.

Also disclosed herein is a compound selected from the compounds in Table1, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.Also disclosed herein is a compound selected from any compound disclosedabove or herein, or a tautomer thereof, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable excipient.

Also disclosed herein is a method of modulating activity of an immunecell, the method comprising contacting the immune cell with an effectiveamount of any compound disclosed above or herein, or a tautomer thereof,or a pharmaceutically acceptable salt thereof.

Also disclosed herein is a method of treating a cancer responsive toinhibition of Cbl-b activity in an individual in need thereof, themethod comprising administering an effective amount of any compounddisclosed above or herein, or a tautomer thereof, or a pharmaceuticallyacceptable salt thereof, to the individual.

Also disclosed herein is a method of inhibiting Cbl-b activity in anindividual in need thereof, the method comprising administering aneffective amount of any compound disclosed above or herein, or atautomer thereof, or a pharmaceutically acceptable salt thereof, to theindividual.

Also disclosed herein is a method for treating or preventing a diseaseor condition associated with Cbl-b activity in an individual in needthereof, the method comprising administering any compound disclosedabove or herein, or a tautomer thereof, or a pharmaceutically acceptablesalt thereof, to the individual.

Also disclosed herein is a method of producing a modified immune cell,the method comprising culturing a cell population containing an immunecell in the presence of an effective amount of any compound disclosedabove or herein, or a tautomer thereof, or a pharmaceutically acceptablesalt thereof.

Also disclosed herein is a modified immune cell comprising a Cbl-binhibitor, wherein the Cbl-b inhibitor is any compound disclosed aboveor herein, or a tautomer thereof, or a pharmaceutically acceptable saltthereof.

Also disclosed herein is an isolated modified immune cell, wherein theimmune cell has been contacted or is in contact with any compounddisclosed above or herein, or a tautomer thereof, or a pharmaceuticallyacceptable salt thereof.

Also disclosed herein is a composition comprising a cell populationcontaining an isolated modified immune cell, wherein the immune cell hasbeen contacted or is in contact with any compound disclosed above orherein, or a tautomer thereof, or a pharmaceutically acceptable saltthereof.

Also disclosed herein is a method of inhibiting abnormal cellproliferation, the method comprising administering an effective amountof an isolated modified immune cell, wherein the immune cell has beencontacted or is in contact with any compound disclosed above or herein,or a tautomer thereof, or a pharmaceutically acceptable salt thereof, toan individual in need thereof.

Also disclosed herein is a method of inhibiting abnormal cellproliferation, the method comprising administering a compositioncomprising a cell population containing an isolated modified immunecell, wherein the immune cell has been contacted or is in contact withany compound disclosed above or herein, or a tautomer thereof, or apharmaceutically acceptable salt thereof, to an individual in needthereof.

Also disclosed herein is a method of inhibiting abnormal cellproliferation, the method comprising administering an effective amountof any compound disclosed above or herein, or a tautomer thereof, or apharmaceutically acceptable salt thereof.

Also disclosed herein is a cell culture composition comprising a cellpopulation containing an immune cell and a Cbl-b inhibitor, wherein theCbl-b inhibitor is any compound disclosed above or herein, or a tautomerthereof, or a pharmaceutically acceptable salt thereof.

Also disclosed herein is a pharmaceutical composition comprising a Cbl-binhibitor and one or both of an adjuvant and an antigen, wherein theCbl-b inhibitor is any compound disclosed above or herein, or a tautomerthereof, or a pharmaceutically acceptable salt thereof.

Also disclosed herein is an article of manufacture comprising anymodified immune cell as disclosed herein, any composition comprising acell population as disclosed herein, any cell culture composition asdisclosed herein, or any pharmaceutical composition as disclosed herein.

Also disclosed herein is a kit comprising any modified immune cell asdisclosed herein or any composition comprising a cell population asdisclosed herein.

Also disclosed herein is the use of a Cbl-b inhibitor in the manufactureof a medicament for treating or preventing a disease or conditionassociated with Cbl-b activity, wherein the Cbl-b inhibitor is anycompound disclosed above or herein, or a tautomer thereof, or apharmaceutically acceptable salt thereof.

In any of the embodiments disclosed herein, the Cbl-b protein can be amammalian Cbl-b, or a human Cbl-b.

Also, disclosed herein are Cbl-b inhibitor compounds, vaccines, andcompositions comprising Cbl-b inhibitors and vaccines, as well asmethods of use thereof in treating cancer. The Cbl-b inhibitor and thecancer vaccine may be administered to an individual with cancer.

Also, disclosed herein are Cbl-b inhibitor compounds, oncolytic viruses,and compositions comprising Cbl-b inhibitors and oncolytic viruses, aswell as methods of use thereof in treating cancer. The Cbl-b inhibitorand the oncolytic virus may be administered to an individual withcancer.

Provided herein is a method of immunizing, the method comprisingadministering to an individual in need thereof an effective amount of asmall molecule Cbl-b inhibitor, and administering to the individual aneffective amount of a vaccine.

Provided herein is a method of treating cancer, the method comprisingadministering to an individual with cancer an effective amount of asmall molecule Cbl-b inhibitor, and administering to the individual aneffective amount of an oncolytic virus.

Provided herein is a method of treating cancer, the method comprisingadministering to an individual with cancer an effective amount of anagent capable of lowering activation threshold of an immune cell, andadministering to the individual an effective amount of a therapeuticcancer vaccine; or administering to the individual an effective amountof an oncolytic virus.

Provided herein is a pharmaceutical composition comprising a cancervaccine and a small molecule Cbl-b inhibitor, optionally wherein thecomposition further comprises a pharmaceutically acceptable excipient.

Provided herein is a kit for treating cancer, the kit comprising (a) asmall molecule Cbl-b inhibitor; (b) a therapeutic cancer vaccine; and(c) instructions for administration of an effective amount of the Cbl-binhibitor and the therapeutic cancer vaccine to treat cancer in anindividual.

Provided herein is a kit for treating cancer, the kit comprising (a) apharmaceutical composition comprising a small molecule Cbl-b inhibitorand a therapeutic cancer vaccine; and (b) instructions foradministration of an effective amount of the pharmaceutical compositioncomprising the Cbl-b inhibitor and the therapeutic cancer vaccine totreat cancer in an individual.

Provided herein is a pharmaceutical composition comprising an oncolyticvirus and a small molecule Cbl-b inhibitor, optionally wherein thecomposition further comprises a pharmaceutically acceptable excipient.

Provided herein is a kit for treating cancer, the kit comprising (a) asmall molecule Cbl-b inhibitor; (b) an oncolytic virus; and (c)instructions for administration of an effective amount of the Cbl-binhibitor and the oncolytic virus to treat cancer in an individual.

And, provided herein is a kit for treating cancer, the kit comprising(a) a pharmaceutical composition comprising a small molecule Cbl-binhibitor and an oncolytic virus; and (b) instructions foradministration of an effective amount of the pharmaceutical compositioncomprising the small molecule Cbl-b inhibitor and the oncolytic virus totreat cancer in an individual.

BRIEF DESCRIPTION OF THE DRAWING

FIG. A shows tumor volume of individual mice.

FIG. B shows percent conditional survival of mice depicted by theKaplan-Meier plot.

DETAILED DESCRIPTION

Provided herein are compounds and pharmaceutical compositions thatinhibit the Cbl-b enzyme, as well as methods of treatment using suchcompounds and pharmaceutical compositions. The compounds andcompositions can be used in methods of modulating the immune system, fortreatment of diseases, and for treatment of cells in vivo, in vitro, orex vivo. Also, provided herein are pharmaceutical compositionscomprising cancer vaccines and compounds that inhibit the Cbl-b enzyme,as well as methods of treatment using such compounds, cancer vaccines,and pharmaceutical compositions. Also provided herein are pharmaceuticalcompositions comprising oncolytic viruses and compounds that inhibit theCbl-b enzyme, as well as methods of treatment using such compounds,oncolytic viruses, and pharmaceutical compositions.

T-cell activation and T-cell tolerance are tightly controlled processesregulating the immune response to tumors while preventing autoimmunity.Tolerance prevents the immune system from attacking cells expressing“self” antigens. During peripheral tolerance, T-cells that recognize“self” antigens (i.e., self-reactive T-cells) become functionallyunresponsive or are deleted after encountering “self” antigens outsideof the thymus. Peripheral tolerance processes therefore are importantfor preventing autoimmune diseases. Normally, cancer cells are removedby activated T-cells that recognize tumor antigens expressed on thesurface of the cancer cells. However, in cancer, the tumormicroenvironment can support T-cell tolerance to cancer cells, whichallows cancer cells to avoid recognition and removal by the immunesystem. The ability of cancer cells to avoid tumor immunosurveillancecan contribute to uncontrolled tumor growth. Therefore, T-cell tolerancecan be a form of T-cell dysfunction. General principles of T-celldysfunction are well known in the art (see Schietinger et al., TrendsImmunol., 35: 51-60, 2014). Additional types of T-cell dysfunction thatcan contribute to uncontrolled tumor growth include T-cell exhaustion,T-cell senescence, and/or T-cell anergy. Therefore, treating T-celldysfunction, for example, by increasing T-cell activation, increasingT-cell proliferation, decreasing T-cell tolerance, and/or decreasingT-cell exhaustion, is beneficial for preventing or treating cancer.Additional cells of the immune system are important for recognition andremoval of cancer cells during immune surveillance. For example, NaturalKiller (NK) cells are lymphocytes of the innate immune system that areable to identify and kill cancer cells (see Martinez-Losato et al., ClinCancer Res., 21: 5048-5056, 2015). Recent studies have also shown thatB-cell subsets with distinct phenotypes and functions exhibit diverseroles in the anti-tumor response (see Saravaria et al., Cell MolImmunol., 14: 662-674, 2017). Due to their role in tumor surveillance,NK-cells and B-cells may also be amenable as therapeutic targets for theprevention or treatment of cancer.

Cbl-b is a RING-type E3 ligase that plays an important role in theimmune system due to its function as a negative regulator of immuneactivation. Cbl-b has an essential role in decreasing the activation ofT-cells, thereby enhancing T-cell tolerance. Studies have found thatcbl-b-deficient T-cells display lower thresholds for activation byantigen recognition receptors and co-stimulatory molecules (e.g., CD28).For example, loss of Cbl-b in T-cells uncouples the requirement for CD28costimulation during T-cell activation and proliferation (see Bachmaieret al., Nature, 403: 211-216, 2000). Such cbl-b−/− T-cells are largelyresistant to T-cell anergy, a tolerance mechanism in which T-cells arefunctionally inactivated and T-cell proliferation is greatly impaired(see Jeon et al., Immunity, 21: 167-177, 2004; and Schwartz et al., AnnuRev Immunol., 21: 305-34, 2003). In support of this, loss of Cbl-b incbl-b knockout mice resulted in impaired induction of T-cell toleranceand exacerbated autoimmunity (see Jeon et al., Immunity, 21: 167-177,2004). Importantly, loss of Cbl-b in mice also resulted in a robustanti-tumor response that depends primarily on cytotoxic T-cells. Onestudy showed that cbl-b−/− CD8+ T-cells are resistant to T regulatorycell-mediated suppression and exhibit enhanced activation and tumorinfiltration. Therapeutic transfer of naive cbl-b−/− CD8+ T-cells wassufficient to mediate rejection of established tumors (see Loeser etal., J Exp Med., 204: 879-891, 2007). Recent studies have shown thatCbl-b also plays a role in NK-cell activation. Genetic deletion of Cbl-bor targeted inactivation of its E3 ligase activity allowed NK-cells tospontaneously reject metastatic tumors in a mouse model (see Paolino etal., Nature, 507: 508-512, 2014).

Provided herein are compounds and compositions that are potentinhibitors of Cbl-b and can be used in novel approaches to treatdiseases such as cancer. In some embodiments, the compounds andcompositions provided herein can be used in methods of modulating theimmune system, such as increasing activation of T-cells, NK-cells, andB-cells, as well as in the treatment of such cells in vivo, in vitro, orex vivo.

I. DEFINITIONS

An “effective amount” of an agent disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” may be determined empirically and in a routine manner, inrelation to the stated purpose. An “effective amount” or an “amountsufficient” of an agent is that amount adequate to produce a desiredbiological effect, such as a beneficial result, including a beneficialclinical result. In some embodiments, the term “effective amount” refersto an amount of an agent effective to “treat” a disease or disorder inan individual (e.g., a mammal such as a human).

The term “Cbl-b” as used herein refers to a Cbl-b protein. The term alsoincludes naturally occurring variants of Cbl-b, including splicevariants or allelic variants. The term also includes non-naturallyoccurring variants of Cbl-b, such as a recombinant Cbl-b protein ortruncated variants thereof, which generally preserve the binding abilityof naturally occurring Cbl-b or naturally occurring variants of Cbl-b(e.g., the ability to bind to an E2 enzyme).

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to preparations that are in such form as to permit the biologicalactivity of the active ingredient to be effective, and that contain noadditional components that are unacceptably toxic to an individual towhich the formulation or composition would be administered. Suchformulations or compositions may be sterile. Such formulations orcompositions may be sterile, with the exception of the inclusion of anoncolytic virus.

“Excipients” as used herein include pharmaceutically acceptableexcipients, carriers, vehicles, or stabilizers that are nontoxic to thecell or mammal being exposed thereto at the dosages and concentrationsemployed. Often the physiologically acceptable excipient is an aqueouspH buffered solution.

Reference to a compound as described in a pharmaceutical composition, orto a compound as described in a claim to a pharmaceutical composition,refers to the compound described by the formula recited in thepharmaceutical composition, without the other elements of thepharmaceutical composition, that is, without carriers, excipients, etc.

The terms “treating” or “treatment” of a disease refer to executing aprotocol, which may include administering one or more therapeutic agentto an individual (human or otherwise), in an effort to obtain beneficialor desired results in the individual, including clinical results.Beneficial or desired clinical results include, but are not limited to,alleviation or amelioration of one or more symptoms, diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total). “Treatment” also can mean prolonging survival ascompared to expected survival of an individual not receiving treatment.Further, “treating” and “treatment” may occur by administration of onedose of a therapeutic agent or therapeutic agents, or may occur uponadministration of a series of doses of a therapeutic agent ortherapeutic agents. “Treating” or “treatment” does not require completealleviation of signs or symptoms, and does not require a cure.“Treatment” also can refer to clinical intervention, such asadministering one or more therapeutic agents to an individual, designedto alter the natural course of the individual or cell being treated(i.e., to alter the course of the individual or cell that would occur inthe absence of the clinical intervention). The term “therapeutic agent”can refer to a Cbl-b inhibitor, a modified immune cell or compositionsthereof.

As used herein, an “individual” or a “subject” is a mammal. A “mammal”for purposes of treatment includes humans; non-human primates; domesticand farm animals; and zoo, sports, or pet animals, such as dogs, horses,rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats,etc. In some embodiments, the individual or subject is human.

As used herein, the term “T-cell dysfunction” refers to a state ofreduced immune responsiveness to antigenic stimulation. The term “T-celldysfunction” includes common elements of both T-cell exhaustion and/orT-cell anergy in which antigen recognition may occur, but the ensuingimmune response is ineffective to control tumor growth. The term “T-celldysfunction” also includes being refractory or unresponsive to antigenrecognition, such as, impaired capacity to translate antigen recognitionto downstream T-cell effector functions, such as proliferation, cytokineproduction, and/or target cell killing.

The term “T-cell anergy” refers to the state of unresponsiveness toantigen stimulation resulting from incomplete or insufficient signalsdelivered through the T-cell receptor. “T-cell anergy” can also resultupon stimulation with antigen in the absence of co-stimulation,resulting in the cell becoming refractory to subsequent activation bythe antigen even in the context of co-stimulation.

The term “T-cell exhaustion” refers to a state of T-cell dysfunctionthat arises from sustained TCR signaling that can occur during cancer.It is distinguished from anergy in that it arises not through incompleteor deficient signaling, but from sustained signaling. It is defined bypoor effector function, sustained expression of inhibitory receptors,and a transcriptional state distinct from that of functional effector ormemory T-cell.

A “T-cell dysfunction disorder” is a disorder or condition characterizedby decreased responsiveness of T-cells to antigenic stimulation.Decreased responsiveness may result in ineffective control of a tumor.In some embodiments, the term “T-cell dysfunction disorder” encompassescancer such as a hematologic cancer or a non-hematologic cancer. In someembodiments, a “T-cell dysfunctional disorder” is one in which T-cellsare anergic or have decreased ability to secrete cytokines, proliferate,or execute cytolytic activity.

“Enhancing T-cell function” means to induce, cause, or stimulate aT-cell to have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhanced T-cellfunction include increased T-cell activation (e.g., increased cytokineproduction, increased expression of T-cell activation markers, etc.),increased T-cell proliferation, decreased T-cell exhaustion, and/ordecreased T-cell tolerance relative to the state of the T-cells beforetreatment with a Cbl-b inhibitor. Methods of measuring enhancement ofT-cell function are known in the art.

“Proliferation” is used herein to refer to the proliferation of a cell.Increased proliferation encompasses the production of a greater numberof cells relative to a baseline value. Decreased proliferationencompasses the production of a reduced number of cells relative to abaseline value. In some embodiments, the cell is an immune cell such asa T-cell and increased proliferation is desired. In some embodiments,the cell is a cancer cell and reduced proliferation is desired.

“Alkyl” as used herein refers to a saturated linear (i.e., unbranched)or branched univalent hydrocarbon chain or combination thereof.Particular alkyl groups are those having a designated number of carbonatoms, for example, an alkyl group having 1 to 20 carbon atoms (a“C₁-C₂₀ alkyl”), having 1 to 10 carbon atoms (a “C₁-C₁₀” alkyl), having1 to 8 carbon atoms (a “C₁-C₈ alkyl”), having 1 to 6 carbon atoms (a“C₁-C₆ alkyl”), having 2 to 6 carbon atoms (a “C₂-C₆ alkyl”), or having1 to 4 carbon atoms (a “C₁-C₄ alkyl”). Examples of alkyl groups include,but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomersof, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

“Alkenyl” as used herein refers to an unsaturated linear (i.e.,unbranched) or branched univalent hydrocarbon chain or combinationthereof, having at least one site of olefinic unsaturation (i.e., havingat least one moiety of the formula C═C). Particular alkenyl groups arethose having a designated number of carbon atoms, for example, analkenyl group having 2 to 20 carbon atoms (a “C₂-C₂₀ alkenyl”), having 2to 10 carbon atoms (a “C₂-C₁₀” alkenyl), having 2 to 8 carbon atoms (a“C₂-C₈ alkenyl”), having 2 to 6 carbon atoms (a “C₂-C₆ alkenyl”), orhaving 2 to 4 carbon atoms (a “C2-C4 alkenyl”). The alkenyl group may bein “cis” or “trans” configurations or, alternatively, in “E” or “Z”configurations. Examples of alkenyl groups include, but are not limitedto, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (orallyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl,buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs, and isomers thereof,and the like.

“Alkynyl” as used herein refers to an unsaturated linear (i.e.,unbranched) or branched univalent hydrocarbon chain or combinationthereof, having at least one site of acetylenic unsaturation (i.e.,having at least one moiety of the formula C≡C). Particular alkynylgroups are those having a designated number of carbon atoms, forexample, an alkynyl group having 2 to 20 carbon atoms (a “C₂-C₂₀alkynyl”), having 2 to 10 carbon atoms (a “C₂-C₁₀ alkynyl”), having 2 to8 carbon atoms (a “C₂-C₈ alkynyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkynyl”), or having 2 to 4 carbon atoms (a “C₂-C₄ alkynyl”). Examplesof alkynyl groups include, but are not limited to, groups such asethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl),but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs, and isomers thereof, andthe like.

“Alkylene” as used herein refers to the same residues as alkyl, buthaving bivalency. Particular alkylene groups are those having 1 to 6carbon atoms (a “C₁-C₆ alkylene”), 1 to 5 carbon atoms (a “C₁-C₅alkylene”), 1 to 4 carbon atoms (a “C₁-C₄ alkylene”), or 1 to 3 carbonatoms (a “C₁-C₃ alkylene”). Examples of alkylene groups include, but arenot limited to, groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), butylene (—CH₂CH₂CH₂CH₂—), and the like.

“Cycloalkyl” as used herein refers to non-aromatic, saturated orunsaturated, cyclic univalent hydrocarbon structures. Particularcycloalkyl groups are those having a designated number of annular (i.e.,ring) carbon atoms, for example, a cycloalkyl group having from 3 to 12annular carbon atoms (a “C₃-C₁₂ cycloalkyl”). A particular cycloalkyl isa cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a “C₃-C₈cycloalkyl”), or having 3 to 6 annular carbon atoms (a “C₃-C₆cycloalkyl”). Cycloalkyl can consist of one ring, such as cyclohexyl, ormultiple rings, such as adamantyl, but excludes aromatic (e.g., aryl)groups. A cycloalkyl comprising more than one ring may be fused, spiro,or bridged, or combinations thereof. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl

cyclobutyl,

and the like.

“Cycloalkylene” as used herein refers to the same residues ascycloalkyl, but having bivalency (i.e., in contrast to being univalent).Particular cycloalkylene groups are those having 3 to 12 annular carbonatoms (a “C₃-C₁₂ cycloalkylene”), having from 3 to 8 annular carbonatoms (a “C₃-C₈ cycloalkylene”), or having 3 to 6 annular carbon atoms(a “C₃-C₆ cycloalkylene”). Examples of cycloalkylene groups include, butare not limited to,

1,2-cyclohexenylene, 1,3-cyclohexenylene, 1,4-cyclohexenylene,cycloheptylene

norbornylene, and the like.

“Aryl” as used herein refers to an aromatic carbocyclic group having asingle ring (e.g., phenyl), or multiple condensed rings (e.g., naphthylor anthryl) where one or more of the condensed rings may not bearomatic. Particular aryl groups are those having from 6 to 14 annular(i.e., ring) carbon atoms (a “C₆-C₁₄ aryl”). An aryl group having morethan one ring where at least one ring is non-aromatic may be connectedto the parent structure at either an aromatic ring position or at anon-aromatic ring position. In one variation, an aryl group having morethan one ring where at least one ring is non-aromatic is connected tothe parent structure at an aromatic ring position. Examples of arylsinclude, but are not limited to, groups such as phenyl, naphthyl,1-naphthyl, 2-naphthyl, 1,2,3,4-tetrahydronaphthalen-6-yl

and the like.

“Carbocyclyl” or “carbocyclic” refers to an aromatic or non-aromaticunivalent cyclic group in which all of the ring members are carbonatoms, such as cyclohexyl, phenyl, 1,2-dihydronaphthyl, etc.

“Arylene” as used herein refers to the same residues as aryl, but havingbivalency. Particular arylene groups are those having from 6 to 14annular carbon atoms (a “C₆-C₁₄ arylene”). Examples of arylene include,but are not limited to, groups such as phenylene, o-phenylene (i.e.,1,2-phenylene), m-phenylene (i.e., 1,3-phenylene), p-phenylene (i.e.,1,4-phenylene), naphthylene, 1,2-naphthylene, 1,3-naphthylene,1,4-naphthylene, 2,7-naphthylene, 2,6-naphthylene, and the like.

“Heteroaryl” as used herein refers to an unsaturated aromatic cyclicgroup having from 1 to 14 annular carbon atoms and at least one annularheteroatom, including, but not limited to, heteroatoms such as nitrogen,oxygen, and sulfur. A heteroaryl group may have a single ring (e.g.,pyridyl or imidazolyl) or multiple condensed rings (e.g., indolizinyl,indolyl, or quinolinyl) where at least one of the condensed rings isaromatic. Particular heteroaryl groups are 5- to 14-membered ringshaving 1 to 12 annular carbon atoms and 1 to 6 annular heteroatomsindependently selected from the group consisting of nitrogen (N), oxygen(O), and sulfur (S) (a “5- to 14-membered heteroaryl”); 5- to10-membered rings having 1 to 8 annular carbon atoms and 1 to 4 annularheteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur (a “5- to 10-membered heteroaryl”); or 5-,6-, or 7-membered rings having 1 to 5 annular carbon atoms and 1 to 4annular heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur (a “5- to 7-membered heteroaryl”). In onevariation, heteroaryl includes monocyclic aromatic 5-, 6-, or 7-memberedrings having from 1 to 6 annular carbon atoms and 1 to 4 annularheteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. In another variation, heteroaryl includespolycyclic aromatic rings having from 1 to 12 annular carbon atoms and 1to 6 annular heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. A heteroaryl group havingmore than one ring where at least one ring is non-aromatic may beconnected to the parent structure at either an aromatic ring position orat a non-aromatic ring position. Examples of heteroaryl include, but arenot limited to, groups such as pyridyl, benzimidazolyl, benzotriazolyl,benzo[b]thienyl, quinolinyl, indolyl, benzothiazolyl, and the like.“Heteroaryl” also includes moieties such as

(2,4-dihydro-3H-1,2,4-triazol-3-one-2-yl), which has the aromatictautomeric structure

(1H-1,2,4-triazol-5-ol-1-yl).

“Heterocyclyl” and “heterocyclic groups” as used herein refer tonon-aromatic saturated or partially unsaturated cyclic groups having thenumber of atoms and heteroatoms as specified, or if no number of atomsor heteroatoms is specified, having at least three annular atoms, from 1to 14 annular carbon atoms, and at least one annular heteroatom,including, but not limited to, heteroatoms such as nitrogen, oxygen, andsulfur. A heterocyclic group may have a single ring (e.g.,tetrahydrothiophenyl, oxazolidinyl) or multiple condensed rings (e.g.,decahydroquinolinyl, octahydrobenzo[d]oxazolyl). Multiple condensedrings include, but are not limited to, bicyclic, tricyclic, andquadracylic rings, as well as bridged or spirocyclic ring systems.Examples of heterocyclic groups include, but are not limited to,aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, oxazolidinyl, piperazinyl,morpholinyl, dioxanyl, 3,6-dihydro-2H-pyranyl,2,3-dihydro-1H-imidazolyl, and the like.

“Heteroarylene” as used herein refers to the same residues asheteroaryl, but having bivalency. Particular heteroarylene groups are 5-to 14-membered rings having 1 to 12 annular carbon atoms and 1 to 6annular heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur (a “5- to 14-membered heteroarylene”); 5-to 10-membered rings having 1 to 8 annular carbon atoms and 1 to 4annular heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur (a “5- to 10-membered heteroarylene”); or5-, 6-, or 7-membered rings having 1 to 5 annular carbon atoms and 1 to4 annular heteroatoms independently selected from the group consistingof nitrogen, oxygen, and sulfur (a “5- to 7-membered heteroarylene”).Examples of heteroarylene include, but are not limited to, groups suchas pyridylene, benzimidazolylene, benzotriazolylene, benzo[b]thienylene,quinolinylene, indolylene, benzothiazolylene, and the like.

“Halo” or “halogen” refers to elements of the Group 17 series havingatomic number 9 to 85. Halo groups include fluoro (F), chloro (Cl),bromo (Br), and iodo (I).

“Haloalkyl,” “haloalkylene,” “haloaryl,” “haloarylene,”“haloheteroaryl,” and similar terms refer to a moiety substituted withat least one halo group. Where a haloalkyl moiety or otherhalo-substituted moiety is substituted with more than one halogen, itmay be referred to by using a prefix corresponding to the number ofhalogen moieties attached. For example, dihaloaryl, dihaloalkyl,trihaloaryl, trihaloalkyl, etc., refer to aryl and alkyl substitutedwith two (“di”) or three (“tri”) halo groups, which may be, but are notnecessarily, the same halo; thus, for example, the haloaryl group4-chloro-3-fluorophenyl is within the scope of dihaloaryl. The subset ofhaloalkyl groups in which each hydrogen (H) of an alkyl group isreplaced with a halo group is referred to as a “perhaloalkyl.” Aparticular perhaloalkyl group is trifluoroalkyl (—CF₃). Similarly,“perhaloalkoxy” refers to an alkoxy group in which a halogen takes theplace of each hydrogen (H) in the hydrocarbon making up the alkyl moietyof the alkoxy group. An example of a perhaloalkoxy group istrifluoromethoxy (—OCF₃). “Haloalkyl” includes monohaloalkyl,dihaloalkyl, trihaloalkyl, perhaloalkyl, and any other number of halosubstituents possible on an alkyl group; and similarly for other groupssuch as haloalkylene, haloaryl, haloarylene, haloheteroaryl, etc.

“Amino” refers to the group —NH₂.

“Oxo” refers to the group ═O, that is, an oxygen atom doubly bonded tocarbon or another chemical element.

“Optionally substituted,” unless otherwise specified, means that a groupis unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4, or 5)of the substituents listed for that group, in which the substituents maybe the same or different. In one embodiment, an optionally substitutedgroup is unsubstituted. In one embodiment, an optionally substitutedgroup has one substituent. In another embodiment, an optionallysubstituted group has two substituents. In another embodiment, anoptionally substituted group has three substituents. In anotherembodiment, an optionally substituted group has four substituents. Insome embodiments, an optionally substituted group has 1 to 2, 1 to 3, 1to 4, or 1 to 5 substituents. When multiple substituents are present,each substituent is independently chosen unless indicated otherwise. Forexample, each (C₁-C₄ alkyl) substituent on the group —N(C₁-C₄alkyl)(C₁-C₄ alkyl) can be selected independently from the other, so asto generate groups such as —N(CH₃)(CH₂CH₃), etc.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms (H) of the specified group or radical are each,independently of one another, replaced with the same or differentsubstituent groups as defined herein. In some embodiments, a group thatis substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents,1 or 2 substituents, or 1 substituent.

Substituents can be attached to any chemically possible location on thespecified group or radical, unless indicated otherwise. Thus, forexample, in one embodiment, —C₁-C₈ alkyl-OH includes, for example,—CH₂CH₂OH and —CH(OH)—CH₃, and —CH₂C(OH)(CH₃)₂, and the like. By way offurther example, in one embodiment, —C₁-C₆ alkyl-OH includes, forexample, —CH₂CH₂OH and —CH(OH)—CH₃, and —CH₂C(OH)(CH₃)₂, and the like.By way of further example, in one embodiment, —C₁-C₆ alkyl-CN includes,for example, —CH₂CH₂CN and —CH(CN)—CH₃, and —CH₂C(CN)(CH₃)₂, and thelike.

Unless a specific isotope of an element is indicated in a formula, thedisclosure includes all isotopologues of the compounds disclosed herein,such as, for example, deuterated derivatives of the compounds (where Hcan be 2H, i.e., deuterium (D)). Deuterated compounds may providefavorable changes in pharmacokinetic (ADME) properties. Isotopologuescan have isotopic replacements at any or at all locations in astructure, or can have atoms present in natural abundance at any or alllocations in a structure.

A “small molecule” as used herein refers to a compound of 1,000 daltonsor less in molecular weight.

Hydrogen atoms can also be replaced with close bioisosteres, such asfluorine, provided that such replacements result in stable compounds.

The disclosure also includes any or all of the stereochemical forms,including any enantiomeric or diastereomeric forms of the compoundsdescribed herein, and cis/trans or E/Z isomers. Unless stereochemistryis explicitly indicated in a chemical structure or name, the structureor name is intended to embrace all possible stereoisomers of a compounddepicted. In addition, where a specific stereochemical form is depicted,it is understood that all other stereochemical forms are also describedand embraced by the disclosure, as well as the generalnon-stereospecific form and mixtures of the disclosed compounds in anyratio, including mixtures of two or more stereochemical forms of adisclosed compound in any ratio, such that racemic, non-racemic,enantioenriched, and scalemic mixtures of a compound are embraced.Compositions comprising a disclosed compound also are intended, such asa composition of substantially pure compound, including a specificstereochemical form thereof. Compositions comprising a mixture ofdisclosed compounds in any ratio also are embraced by the disclosure,including compositions comprising mixtures of two or more stereochemicalforms of a disclosed compound in any ratio, such that racemic,non-racemic, enantioenriched, and scalemic mixtures of a compound areembraced by the disclosure. If stereochemistry is explicitly indicatedfor one portion or portions of a molecule, but not for another portionor portions of a molecule, the structure is intended to embrace allpossible stereoisomers for the portion or portions where stereochemistryis not explicitly indicated. The disclosure also embraces any and alltautomeric forms of the compounds described herein.

The disclosure is intended to embrace all salts of the compoundsdescribed herein, as well as methods of using such salts of thecompounds. In one embodiment, the salts of the compounds comprisepharmaceutically acceptable salts. Pharmaceutically acceptable salts arethose salts that can be administered as drugs or pharmaceuticals tohumans and/or animals and that, upon administration, retain at leastsome of the biological activity of the free compound (neutral compoundor non-salt compound). The desired salt of a basic compound may beprepared by methods known to those of skill in the art by treating thecompound with an acid. Examples of inorganic acids include, but are notlimited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, and phosphoric acid. Examples of organic acids include, but arenot limited to, formic acid, acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, sulfonic acids, and salicylic acid. Salts of basiccompounds with amino acids, such as aspartate salts and glutamate salts,also can be prepared. The desired salt of an acidic compound can beprepared by methods known to those of skill in the art by treating thecompound with a base. Examples of inorganic salts of acid compoundsinclude, but are not limited to, alkali metal and alkaline earth salts,such as sodium salts, potassium salts, magnesium salts, and calciumsalts; ammonium salts; and aluminum salts. Examples of organic salts ofacid compounds include, but are not limited to, procaine, dibenzylamine,N-ethylpiperidine, N,N′-dibenzylethylenediamine, and triethylaminesalts. Salts of acidic compounds with amino acids, such as lysine salts,also can be prepared. For lists of pharmaceutically acceptable salts,see, for example, P. H. Stahl and C. G. Wermuth (eds.) “Handbook ofPharmaceutical Salts, Properties, Selection and Use” Wiley-VCH, 2011(ISBN: 978-3-90639-051-2). Several pharmaceutically acceptable salts arealso disclosed in Berge, J. Pharm. Sci. 66:1 (1977).

As described in Biological Example 1, 8, and/or 12, the Cbl-b activityassay (Cbl-b inhibition assay) used to measure the IC₅₀ values for Cbl-binhibition uses a mixture comprising an N-terminal biotinylatedAvi-tagged Cbl-b, a fluorescently-labeled inhibitor probe tagged withBODIPY FL (Example 46), and assay buffer. In one embodiment, the Cbl-bactivity assay (Cbl-b inhibition assay) used to measure IC₅₀ forinhibition of Cbl-b uses the conditions described in Biological Example1, 8, and/or 12, with 0.5 nM Cbl-b (“High” final concentration). Inanother embodiment, the Cbl-b activity assay (Cbl-b inhibition assay)used to measure IC₅₀ for inhibition of Cbl-b uses the conditionsdescribed in Biological Example 1, 8, and/or 12, with 0.125 nM Cbl-b(“Low” final concentration).

It is appreciated that certain features disclosed herein, which are, forclarity, described in the context of separate embodiments, also may beprovided in combination in a single embodiment. Conversely, variousfeatures disclosed herein, which are, for brevity, described in thecontext of a single embodiment, also may be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables also are specifically embraced by the presentdisclosure and are disclosed herein just as if each and every suchsubcombination of chemical groups was individually and explicitlydisclosed herein.

It is understood that aspects and embodiments described herein as“comprising” include “consisting of” and “consisting essentially of”embodiments.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless otherwise indicated or clearfrom context. For example, “an” excipient includes one or moreexcipients.

Reference to “about” a value, encompasses from 90% to 110% of thatvalue. For instance, about 50 billion cells refers to 45 to 55 billioncells, and includes 50 billion cells. For instance, a temperature of“about 100 degrees” refers to a temperature of about 90 degrees to about110 degrees.

When numerical ranges of compounds are given, all compounds within thosenumerical limits, including any designated “a” and “b,” are included,unless expressly excluded. For example, reference to compounds 41-43refers to compounds 41, 42, and 43.

II. COMPOUNDS

In one aspect, provided is a compound of Formula (I):

-   -   or a tautomer thereof, or a pharmaceutically acceptable salt        thereof,    -   wherein:

is

-   -   X is CH or N;    -   Z¹ is CH or N;    -   Z² is CH or N;    -   R^(1a) and R^(1b) are independently H, C₁-C₆ alkyl, C₁-C₆        haloalkyl, or C₁-C₆ alkyl-OH;    -   R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkyl-OH,        C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl);    -   R^(2b) is H, halo, or C₁-C₆ alkyl;    -   or R^(2a) and R^(2b) are taken together with the carbon atom to        which they are attached to form a spiro 3- to 6-membered        heterocyclyl or a spiro C₃-C₆ cycloalkyl,        -   wherein at least one of the atoms of the spiro heterocyclyl            which is adjacent to the connecting piperidinyl ring is            carbon;    -   R^(3a) and R^(3b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form C₃-C₄ cycloalkyl;    -   R⁴ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH;

is

and Y is CR^(5a)R^(5b) or S;

-   -   or

is

and Y is a bond;

-   -   W is O or a bond;    -   R^(5a) and R^(5b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form a C₃-C₆ cycloalkyl;    -   R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁷ is H, halo, C₃-C₆ cycloalkyl, —NH-(3- to 6-membered        heterocyclyl), —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl), —O-(3-        to 6-membered heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆        cycloalkyl);    -   R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁹ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and    -   R¹⁰ is —CF₃ or cyclopropyl.

In another aspect, provided is a compound of Formula (I-a):

-   -   or a tautomer thereof, or a pharmaceutically acceptable salt        thereof,    -   wherein:

is

-   -   X is CH or N;    -   Z¹ is CH or N;    -   Z² is CH or N;    -   R^(1a) and R^(1b) are independently H, C₁-C₆ alkyl, C₁-C₆        haloalkyl, or C₁-C₆ alkyl-OH;    -   R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkyl-OH,        C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl);    -   R^(2b) is H, halo, or C₁-C₆ alkyl;    -   or R^(2a) and R^(2b) are taken together with the carbon atom to        which they are attached to form a spiro 3- to 6-membered        heterocyclyl or a spiro C₃-C₆ cycloalkyl,        -   wherein at least one of the atoms of the spiro heterocyclyl            which is adjacent to the connecting piperidinyl ring is            carbon;    -   R^(3a) and R^(3b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form C₃-C₄ cycloalkyl;    -   R⁴ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH;

is

and Y is CR^(5a)R^(5b) or S;

-   -   or

is

and Y is a bond;

-   -   W is O or a bond;    -   R^(5a) and R^(5b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form a C₃-C₆ cycloalkyl;    -   R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁷ is H, C₃-C₆ cycloalkyl, —NH-(3- to 6-membered heterocyclyl),        —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl), —O-(3- to 6-membered        heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆ cycloalkyl);    -   R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁹ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and    -   R¹⁰ is —CF₃ or cyclopropyl.

In some embodiments,

(i.e., the Ring A moiety), is

In some embodiments, Z¹ is CH. In other embodiments, Z¹ is N. In someembodiments, the Ring A moiety is

In some embodiments, Z² is CH. In other embodiments, Z² is N. In someembodiments, R¹⁰ is —CF₃. In other embodiments, R¹⁰ is cyclopropyl. Insome embodiments, the Ring A moiety is selected from the groupconsisting of:

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, the Ring A moiety is

In some embodiments, R^(1a) and R^(1b) are independently H, C₁-C₆ alkyl,C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH. In some embodiments, R^(1a) andR^(1b) are independently H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃alkyl-OH. In some embodiments, R^(1a) and R^(1b) are independently H,—CH₃, —CF₃, or —CH₂OH. In some embodiments, R^(1a) is H. In someembodiments, R^(1b) is H. In some embodiments, R^(1a) and R^(1b) areboth H. In some embodiments, one of R^(1a) and R^(1b) is H, and theother is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH.

In some embodiments, R^(1a) and R^(1b) are independently C₁-C₆ alkyl. Insome embodiments, R^(1a) and R^(1b) are independently C₁-C₃ alkyl. Insome embodiments, R^(1a) and R^(1b) are independently methyl, ethyl,n-propyl, or isopropyl. In some embodiments, R^(1a) and R^(1b) are bothmethyl. In some embodiments, one of R^(1a) and R^(1b) is H, and theother is methyl.

In some embodiments, R^(1a) and R^(1b) are independently C₁-C₆haloalkyl. In some embodiments, R^(1a) and R^(1b) are independentlyC₁-C₆ haloalkyl containing 1-13 halogen atoms. In some embodiments,R^(1a) and R^(1b) are independently C₁-C₆ haloalkyl containing 1-7halogen atoms. In some embodiments, R^(1a) and R^(1b) are independentlyC₁-C₃ haloalkyl. In some embodiments, R^(1a) and R^(1b) areindependently C₁-C₃ haloalkyl containing 1-7 halogen atoms. In someembodiments, R^(1a) and R^(1b) are independently C₁-C₂ haloalkylcontaining 1-5 halogen atoms. In some embodiments, the halogen atoms areindependently selected from the group consisting of chloro, bromo, andfluoro atoms. In some embodiments, the halogen atoms are independentlyselected from the group consisting of chloro and fluoro atoms. In someembodiments, the halogen atoms are all fluoro atoms. In someembodiments, the halogen atoms are a combination of chloro and fluoroatoms. In some embodiments, R^(1a) and R^(1b) are independently —CF₃,—CCl₃, —CF₂Cl, —CFCl₂, —CHF₂, —CH₂F, —CHCl₂, —CH₂F, or —CHFCl. In someembodiments, R^(1a) and R^(1b) are both —CF₃. In some embodiments, oneof R^(1a) and R^(1b) is H, and the other is —CF₃.

In some embodiments, R^(1a) and R^(1b) are independently C₁-C₆ alkyl-OH.In some embodiments, R^(1a) and R^(1b) are independently C₁-C₃ alkyl-OH.In some embodiments, R^(1a) and R^(1b) are independently —CH₂OH,—CH₂CH₂—OH, —CH₂CH₂CH₂—OH, or —C(CH₃)₂—OH. In some embodiments, R^(1a)and R^(1b) are both —CH₂OH. In some embodiments, one of R^(1a) andR^(1b) is H, and the other is —CH₂OH.

Compounds of formula (I) contain a carbon atom directly linked to theconnecting piperidinyl ring in at least one of the positionscorresponding to substituents R^(2a) and R^(2b). In some embodiments,compounds of formula (I) contain a carbon atom directly linked to theconnecting piperidinyl ring in both of the positions corresponding tosubstituents R^(2a) and R^(2b).

In some embodiments, R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkyl-OH, C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl). In someembodiments, R^(2a) is —CN, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃alkyl-OH, C₁-C₃ alkyl-CN, or —(C₁-C₃ alkylene)-O—(C₁-C₃ alkyl). In someembodiments, R^(2a) is —CN, —CH₃, —CF₃, —CH₂OH, —CH₂CN, or —CH₂—O—CH₃.

In some embodiments, R^(2a) is —CN.

In some embodiments, R^(2a) is C₁-C₆ alkyl. In some embodiments, R^(2a)is C₁-C₃ alkyl. In some embodiments, R^(2a) is methyl, ethyl, n-propyl,or isopropyl. In some embodiments, R^(2a) is —CH₃.

In some embodiments, R^(2a) is C₁-C₆ haloalkyl. In some embodiments,R^(2a) is C₁-C₆ haloalkyl containing 1-13 halogen atoms. In someembodiments, R^(2a) is C₁-C₆ haloalkyl containing 1-7 halogen atoms. Insome embodiments, R^(2a) is C₁-C₃ haloalkyl. In some embodiments, R^(2a)is C₁-C₃ haloalkyl containing 1-7 halogen atoms. In some embodiments,R^(2a) is C₁-C₂ haloalkyl containing 1-5 halogen atoms. In someembodiments, the halogen atoms are independently selected from the groupconsisting of chloro, bromo, and fluoro atoms. In some embodiments, thehalogen atoms are independently selected from the group consisting ofchloro and fluoro atoms. In some embodiments, the halogen atoms are allfluoro atoms. In some embodiments, the halogen atoms are a combinationof chloro and fluoro atoms. In some embodiments, R^(2a) is —CF₃, —CCl₃,—CF₂Cl, —CFCl₂, —CHF₂, —CH₂F, —CHCl₂, —CH₂F, or —CHFCl. In someembodiments, R^(2a) is —CF₃.

In some embodiments, R^(2a) is C₁-C₆ alkyl-OH. In some embodiments,R^(2a) is C₁-C₃ alkyl-OH. In some embodiments, R^(2a) is —CH₂OH,—CH₂CH₂—OH, —CH₂CH₂CH₂—OH, or —C(CH₃)₂—OH. In some embodiments, R^(2a)is —CH₂OH.

In some embodiments, R^(2a) is C₁-C₆ alkyl-CN. In some embodiments,R^(2a) is C₁-C₃ alkyl-CN. In some embodiments, R^(2a) is —CH₂CN,—CH₂CH₂—CN, —CH₂CH₂CH₂—CN, or —C(CH₃)₂—CN. In some embodiments, R^(2a)is —CH₂CN.

In some embodiments, R^(2a) is —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl). Insome embodiments, R^(2a) is —(C₁-C₃ alkylene)-O—(C₁-C₃ alkyl). In someembodiments, R^(2a)—(C₁-C₂ alkylene)-O—(C₁-C₂ alkyl). In someembodiments, R^(2a) is —CH₂—O—CH₃, —CH₂—O—CH₂CH₃, —CH₂CH₂—O—CH₂CH₃, is—CH₂CH₂—O—CH₃. In some embodiments, R^(2a) is —CH₂—O—CH₃.

In some embodiments, R^(2b) is H, halo, or C₁-C₆ alkyl. In someembodiments, R^(2b) is H, halo, or C₁-C₃ alkyl. In some embodiments,R^(2b) is H, F, or —CH₃.

In some embodiments, R^(2b) is H.

In some embodiments, R^(2b) is halo. In some embodiments, R^(2b) ischloro, fluoro, or bromo. In some embodiments, R^(2b) is chloro orfluoro. In some embodiments, R^(2b) is fluoro.

In some embodiments, R^(2b) is C₁-C₆ alkyl. In some embodiments, R^(2b)is C₁-C₃ alkyl. In some embodiments, R^(2b) is methyl, ethyl, n-propyl,or isopropyl. In some embodiments, R^(2b) is —CH₃.

In some embodiments, R^(2a) and R^(2b) are taken together with thecarbon atom to which they are attached to form a spiro 3- to 6-memberedheterocyclyl or a spiro C₃-C₆ cycloalkyl, wherein at least one of theatoms of the spiro heterocyclyl which is adjacent to the connectingpiperidinyl ring is carbon. In some embodiments, R^(2a) and R^(2b) aretaken together with the carbon atom to which they are attached to form aspiro 4- to 5-membered heterocyclyl or a spiro C₃-C₄ cycloalkyl. In someembodiments, R^(2a) and R^(2b) are taken together with the carbon atomto which they are attached to form a spiro C₃-C₄ cycloalkyl. In someembodiments, R^(2a) and R^(2b) are taken together with the carbon atomto which they are attached to form a spiro cyclopropyl or cyclobutyl. Insome embodiments, R^(2a) and R^(2b) are taken together with the carbonatom to which they are attached to form a spiro cyclopropyl. In someembodiments, R^(2a) and R^(2b) are taken together with the carbon atomto which they are attached to form a spiro 4- to 5-memberedheterocyclyl. In some embodiments, R^(2a) and R^(2b) are taken togetherwith the carbon atom to which they are attached to form a spiro 4- to5-membered heterocyclyl containing 1-3 heteroatoms selected from thegroup consisting of O, N, and S. In some embodiments, R^(2a) and R^(2b)are taken together with the carbon atom to which they are attached toform a spiro 4- to 5-membered heterocyclyl containing 1-3 nitrogenand/or oxygen atoms. In some embodiments, R^(2a) and R^(2b) are takentogether with the carbon atom to which they are attached to form a spiro4- to 5-membered heterocyclyl containing 1-2 oxygen atoms. In someembodiments, R^(2a) and R^(2b) are taken together with the carbon atomto which they are attached to form

In some embodiments, R^(2a) and R^(2b) are taken together with thecarbon atom to which they are attached to form

In some embodiments, R^(2a) and R^(2b) are taken together with thecarbon atom to which they are attached to form

In some embodiments, R^(2a) and R^(2b) are taken together with thecarbon atom to which they are attached to form

In some embodiments, R^(3a) and R^(3b) are independently H, halo, orC₁-C₆ alkyl. In some embodiments, R^(3a) and R^(3b) are independently H,halo, or C₁-C₃ alkyl. In some embodiments, R^(3a) and R^(3b) areindependently H, F, or —CH₃.

In some embodiments, R^(3a) is H. In some embodiments, R^(3b) is H. Insome embodiments, R^(3a) and R^(3b) are both H. In some embodiments, oneof R^(3a) and R^(3b) is H, and the other is halo or C₁-C₆ alkyl.

In some embodiments, R^(3a) and R^(3b) are independently halo. In someembodiments, R^(3a) and R^(3b) are independently chloro, fluoro, orbromo. In some embodiments, R^(3a) and R^(3b) are independently chloroor fluoro. In some embodiments, R^(3a) is fluoro. In some embodiments,R^(3b) is fluoro. In some embodiments, R^(3a) and R^(3b) are both halo.In some embodiments, R^(3a) and R^(3b) are both fluoro.

In some embodiments, R^(3a) and R^(3b) are independently C₁-C₆ alkyl. Insome embodiments, R^(3a) and R^(3b) are independently C₁-C₃ alkyl. Insome embodiments, R^(3a) and R^(3b) are independently methyl, ethyl,n-propyl, or isopropyl. In some embodiments, R^(3a) and R^(3b) are both—CH₃. In some embodiments, one of R^(3a) and R^(3b) is —CH₃.

In some embodiments, R^(3a) and R^(3b) are taken together with thecarbon atom to which they are attached to form C₃-C₄ cycloalkyl. In someembodiments, R^(3a) and R^(3b) are taken together with the carbon atomto which they are attached to form cyclopropyl. In some embodiments,R^(3a) and R^(3b) are taken together with the carbon atom to which theyare attached to form cyclobutyl.

In some embodiments, R⁴ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆alkyl-OH. In some embodiments, R⁴ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, orC₁-C₃ alkyl-OH. In some embodiments, R⁴ is H, —CH₃, —CF₃, or —CH₂OH.

In some embodiments, R⁴ is H.

In some embodiments, R⁴ is C₁-C₆ alkyl. In some embodiments, R⁴ is C₁-C₃alkyl. In some embodiments, R⁴ is methyl, ethyl, n-propyl, or isopropyl.In some embodiments, R⁴ is —CH₃.

In some embodiments, R⁴ is C₁-C₆ haloalkyl. In some embodiments, R⁴ isC₁-C₆ haloalkyl containing 1-13 halogen atoms. In some embodiments, R⁴is C₁-C₆ haloalkyl containing 1-7 halogen atoms. In some embodiments, R⁴is C₁-C₃ haloalkyl. In some embodiments, R⁴ is C₁-C₃ haloalkylcontaining 1-7 halogen atoms. In some embodiments, R⁴ is C₁-C₂ haloalkylcontaining 1-5 halogen atoms. In some embodiments, the halogen atoms areindependently selected from the group consisting of chloro, bromo, andfluoro atoms. In some embodiments, the halogen atoms are independentlyselected from the group consisting of chloro and fluoro atoms. In someembodiments, the halogen atoms are all fluoro atoms. In someembodiments, the halogen atoms are a combination of chloro and fluoroatoms. In some embodiments, R⁴ is —CF₃, —CCl₃, —CF₂Cl, —CFCl₂, —CHF₂,—CH₂F, —CHCl₂, —CH₂F, or —CHFCl. In some embodiments, R⁴ is —CF₃.

In some embodiments, R⁴ is C₁-C₆ alkyl-OH. In some embodiments, R⁴ isC₁-C₃ alkyl-OH. In some embodiments, R⁴ is —CH₂OH, —CH₂CH₂—OH,—CH₂CH₂CH₂—OH, or —C(CH₃)₂—OH. In some embodiments, R⁴ is —CH₂OH.

In some embodiments, X is CH. In other embodiments, X is N.

In some embodiments,

(i.e., the Ring D moiety) is

In some embodiments, W is O and the Ring D moiety is

In other embodiments, W is a bond and the Ring D moiety is

In some embodiments, the Ring D moiety is

In some embodiments, the Ring D moiety is

In some embodiments, the Ring D moiety is

and R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl. In someembodiments, R⁸ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄ cycloalkyl. Insome embodiments, R⁸ is —CH₃, —CF₃, or cyclopropyl. In some embodiments,the Ring D moiety is

In some embodiments, R⁸ is C₁-C₆ alkyl. In some embodiments, R⁸ is C₁-C₃alkyl. In some embodiments, R⁸ is methyl, ethyl, n-propyl, or isopropyl.In some embodiments, R⁸ is —CH₃.

In some embodiments, R⁸ is C₁-C₆ haloalkyl. In some embodiments, R⁸ isC₁-C₆ haloalkyl containing 1-13 halogen atoms. In some embodiments, R⁸is C₁-C₆ haloalkyl containing 1-7 halogen atoms. In some embodiments, R⁸is C₁-C₃ haloalkyl. In some embodiments, R⁸ is C₁-C₃ haloalkylcontaining 1-7 halogen atoms. In some embodiments, R⁸ is C₁-C₂ haloalkylcontaining 1-5 halogen atoms. In some embodiments, the halogen atoms areindependently selected from the group consisting of chloro, bromo, andfluoro atoms. In some embodiments, the halogen atoms are independentlyselected from the group consisting of chloro and fluoro atoms. In someembodiments, the halogen atoms are all fluoro atoms. In someembodiments, the halogen atoms are a combination of chloro and fluoroatoms. In some embodiments, R⁸ is —CF₃, —CCl₃, —CF₂Cl, —CFCl₂, —CHF₂,—CHF, —CHCl₂, —CH₂F, or —CHFCl. In some embodiments, R⁸ is —CF₃.

In some embodiments, R⁸ is C₃-C₄ cycloalkyl. In some embodiments, R⁸ iscyclopropyl. In some embodiments, R⁸ is cyclobutyl.

In some embodiments, Y is S. In other embodiments, Y is a bond. Infurther embodiments, Y is CR^(5a)R^(5b), wherein R^(5a) and R^(5b) areindependently H, halo, or C₁-C₆ alkyl, or R^(5a) and R^(5b) are takentogether with the carbon atom to which they are attached to form a C₃-C₆cycloalkyl.

In some embodiments, R^(5a) and R^(5b) are independently H, halo, orC₁-C₆ alkyl. In some embodiments, R^(5a) and R^(5b) are independently H,halo, or C₁-C₃ alkyl. In some embodiments, R^(5a) and R^(5b) areindependently H, F, or —CH₃.

In some embodiments, R^(5a) is H. In some embodiments, R^(5b) is H. Insome embodiments, R^(5a) and R^(5b) are both H. In some embodiments, oneof R^(5a) and R^(5b) is H, and the other is halo or C₁-C₆ alkyl. In someembodiments, one of R^(5a) and R^(5b) is H, and the other is F or —CH₃.In some embodiments, one of R^(5a) and R^(5b) is H, and the other is F.

In some embodiments, R^(5a) and R^(5b) are independently halo. In someembodiments, R^(5a) and R^(5b) are independently chloro, fluoro, orbromo. In some embodiments, R^(5a) and R^(5b) are independently chloroor fluoro. In some embodiments, R^(5a) and R^(5b) are both halo. In someembodiments, R^(5a) and R^(5b) are both fluoro. In some embodiments, oneof R^(5a) and R^(5b) is fluoro.

In some embodiments, R^(5a) and R^(5b) are taken together with thecarbon atom to which they are attached to form a C₃-C₆ cycloalkyl. Insome embodiments, R^(5a) and R^(5b) are taken together with the carbonatom to which they are attached to form a C₃-C₄ cycloalkyl. In someembodiments, R^(5a) and R^(5b) are taken together with the carbon atomto which they are attached to form cyclopropyl. In some embodiments,R^(5a) and R^(5b) are taken together with the carbon atom to which theyare attached to form cyclobutyl.

In some embodiments, R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆cycloalkyl. In some embodiments, R⁶ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, orC₃-C₄ cycloalkyl. In some embodiments, R⁶ is —CH₃, —CHF₂, orcyclopropyl.

In some embodiments, the Ring D moiety is

and Y is CR^(5a)R^(5b). In some embodiments, the Ring D moiety is

W is O, and Y is CR^(5a)R^(5b). In some embodiments, the Ring D moietyis

W is a bond, and Y is CR^(5a)R^(5b). In some embodiments, the Ring Dmoiety is

and Y is S. In some embodiments, the Ring D moiety is

W is O, and Y is S. In some embodiments, the Ring D moiety is

W is a bond, and Y is S. In some embodiments, the Ring D moiety is

and Y is a bond.

In some embodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₃alkyl. In some embodiments, R⁶ is methyl, ethyl, n-propyl, or isopropyl.In some embodiments, R⁶ is —CH₃.

In some embodiments, R⁶ is C₁-C₆ haloalkyl. In some embodiments, R⁶ isC₁-C₆ haloalkyl containing 1-13 halogen atoms. In some embodiments, R⁶is C₁-C₆ haloalkyl containing 1-7 halogen atoms. In some embodiments, R⁶is C₁-C₃ haloalkyl. In some embodiments, R⁶ is C₁-C₃ haloalkylcontaining 1-7 halogen atoms. In some embodiments, R⁶ is C₁-C₂ haloalkylcontaining 1-5 halogen atoms. In some embodiments, the halogen atoms areindependently selected from the group consisting of chloro, bromo, andfluoro atoms. In some embodiments, the halogen atoms are independentlyselected from the group consisting of chloro and fluoro atoms. In someembodiments, the halogen atoms are all fluoro atoms. In someembodiments, the halogen atoms are a combination of chloro and fluoroatoms. In some embodiments, R⁶ is —CF₃, —CCl₃, —CF₂Cl, —CFCl₂, —CHF₂,—CH₂F, —CHCl₂, —CH₂F, or —CHFCl. In some embodiments, R⁶ is —CHF₂.

In some embodiments, R⁶ is C₃-C₆ cycloalkyl. In some embodiments, R⁶ isC₃-C₅ cycloalkyl. In some embodiments, R⁶ is C₃-C₄ cycloalkyl. In someembodiments, R⁶ is cyclopropyl, cyclobutyl, or cyclopentyl. In someembodiments, R⁶ is cyclopropyl. In some embodiments, R⁶ is cyclobutyl.

In some embodiments, R⁷ is H, halo, C₃-C₆ cycloalkyl, —NH-(3- to6-membered heterocyclyl), —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl),—O-(3- to 6-membered heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆cycloalkyl). In some embodiments, R⁷ is H, halo, C₃-C₄ cycloalkyl,—NH(4- to 5-membered heterocyclyl), —NH(C₁-C₃ alkyl), —NH(C₃-C₅cycloalkyl), —O(C₁-C₃ alkyl), —O(4- to 5-membered heterocyclyl), or—O(C₃-C₅ cycloalkyl). In some embodiments, R⁷ is H, Cl, cyclopropyl,—NH(CH₂CH₃), —NH(cyclopropyl), —OCH₂CH₃, —O(cyclopropyl),

In some embodiments, R⁷ is H.

In some embodiments, R⁷ is halo. In some embodiments, R⁷ is chloro,fluoro, or bromo. In some embodiments, R⁷ is chloro or fluoro. In someembodiments, R⁷ is chloro.

In some embodiments, R⁷ is C₃-C₆ cycloalkyl. In some embodiments, R⁷ isC₃-C₄ cycloalkyl. In some embodiments, R⁷ is cyclopropyl or cyclobutyl.In some embodiments, R⁷ is cyclopropyl.

In some embodiments, R⁷ is —NH-(3- to 6-membered heterocyclyl). In someembodiments, R⁷ is —NH(4- to 5-membered heterocyclyl). In someembodiments, the heterocyclyl moiety of R⁷ contains 1-2 heteroatomsindependently selected from the group consisting of nitrogen and oxygen.In some embodiments, the heterocyclyl moiety of R⁷ contains one oxygenatom. In some embodiments, the heterocyclyl moiety of R⁷ contains onenitrogen atom. In some embodiments, the heterocyclyl moiety of R⁷ isselected from the group consisting of:

In some embodiments, R⁷ is

In some embodiments, R⁷ is —NH—(C₁-C₆ alkyl). In some embodiments, R⁷ is—NH—(C₁-C₃ alkyl). In some embodiments, R⁷ is —N(H)CH₃, —NH(CH₂CH₃),—NH(CH₂CH₂CH₃), or —N(H)CH(CH₃)₂. In some embodiments, R⁷ is—NH(CH₂CH₃).

In some embodiments, R⁷ is —NH—(C₃-C₆ cycloalkyl). In some embodiments,R⁷ is —NH—(C₃-C₅ cycloalkyl). In some embodiments, R⁷ is —NH—(C₃-C₄cycloalkyl). In some embodiments, R⁷ is —NH-(cyclopropyl),—NH-(cyclobutyl), —NH-(cyclopentyl), or —NH-(cyclohexyl). In someembodiments, R⁷ is —NH-(cyclopropyl).

In some embodiments, R⁷ is —O-(3- to 6-membered heterocyclyl). In someembodiments, R⁷ is —O-(4- to 5-membered heterocyclyl). In someembodiments, the heterocyclyl moiety of R⁷ contains 1-2 heteroatomsindependently selected from the group consisting of nitrogen and oxygen.In some embodiments, the heterocyclyl moiety of R⁷ contains one oxygenatom. In some embodiments, the heterocyclyl moiety of R⁷ contains onenitrogen atom. In some embodiments, the heterocyclyl moiety of R⁷ isselected from the group consisting of:

In some embodiments, R⁷ is

In some embodiments, R⁷ is —O—(C₁-C₆ alkyl). In some embodiments, R⁷ is—O—(C₁-C₃ alkyl). In some embodiments, R⁷ is —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, or —OCH(CH₃)₂. In some embodiments, R⁷ is —OCH₂CH₃.

In some embodiments, R⁷ is —O—(C₃-C₆ cycloalkyl). In some embodiments,R⁷ is —O—(C₃-C₅ cycloalkyl). In some embodiments, R⁷ is —O—(C₃-C₄cycloalkyl). In some embodiments, R⁷ is —O-(cyclopropyl),—O-(cyclobutyl), —O-(cyclopentyl), or —O-(cyclohexyl). In someembodiments, R⁷ is —O-(cyclopropyl).

In some embodiments, R⁹ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆alkyl-OH. In some embodiments, R⁹ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, orC₁-C₃ alkyl-OH. In some embodiments, R⁹ is H, —CH₃, —CF₃, or —CH₂OH.

In some embodiments, R⁹ is H.

In some embodiments, R⁹ is C₁-C₆ alkyl. In some embodiments, R⁹ is C₁-C₃alkyl. In some embodiments, R⁹ is methyl, ethyl, n-propyl, or isopropyl.In some embodiments, R⁹ is —CH₃.

In some embodiments, R⁹ is C₁-C₆ haloalkyl. In some embodiments, R⁹ isC₁-C₆ haloalkyl containing 1-13 halogen atoms. In some embodiments, R⁹is C₁-C₆ haloalkyl containing 1-7 halogen atoms. In some embodiments, R⁹is C₁-C₃ haloalkyl. In some embodiments, R⁹ is C₁-C₃ haloalkylcontaining 1-7 halogen atoms. In some embodiments, R⁹ is C₁-C₂ haloalkylcontaining 1-5 halogen atoms. In some embodiments, the halogen atoms areindependently selected from the group consisting of chloro, bromo, andfluoro atoms. In some embodiments, the halogen atoms are independentlyselected from the group consisting of chloro and fluoro atoms. In someembodiments, the halogen atoms are all fluoro atoms. In someembodiments, the halogen atoms are a combination of chloro and fluoroatoms. In some embodiments, R⁹ is —CF₃, —CCl₃, —CF₂Cl, —CFCl₂, —CHF₂,—CH₂F, —CHCl₂, —CH₂F, or —CHFCl. In some embodiments, R⁹ is —CF₃.

In some embodiments, R⁹ is C₁-C₆ alkyl-OH. In some embodiments, R⁹ isC₁-C₃ alkyl-OH. In some embodiments, R⁹ is —CH₂OH, —CH₂CH₂—OH,—CH₂CH₂CH₂—OH, or —C(CH₃)₂—OH. In some embodiments, R⁹ is —CH₂OH.

In some embodiments, the compound is of formula (I-A) or (I-B):

-   -   wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R⁶,        R⁷, R⁹, R¹⁰, X, Y, Z¹, Z², and the Ring D moiety are as        described for the compound of formula (I).

In some embodiments, the compound is of formula (I-C), (I-D), (I-E), or(I-F):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R⁶, R⁷, R⁹,R⁰, W, X, Y, Z¹, and Z² are as defined for the compound of formula (I).

In some embodiments, the compound is of formula (I-G), (I-H), (I-I),(I-J), (I-K), (I-L), (I-M), or (I-N):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R⁶, R⁷, R⁹,R¹⁰, W, X, and Y are as defined for the compound of formula (I).

In some embodiments, the compound is of formula (II-A), (II-B), (II-C),(II-D), (II-E), (II-F), (II-G), (II-H), (II-I), (II-J), (II-K), or(II-L).

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R^(5a),R^(5b), R⁶, R⁷, R⁸, and R⁹ are as defined for the compound of formula(I), and p of formula (II-G) is 1 or 2.

In some embodiments, the compound is of formula (III-A), (III-B),(III-C), or (III-D):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R^(5a),R^(5b), R⁷, and R⁹ are as defined for the compound of formula (I).

In some embodiments, the compound is of formula (IV-A), (IV-B), or(IV-C):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R^(5a),R^(5b), R⁶, R⁷, and R⁹ are as defined for the compound of formula (I).

In some embodiments, the compound is of formula (V-A), (V-B), (V-C),(V-D), (V-E), (V-F), or (V-G):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R^(5a),R^(5b), R⁶, R⁷, and R⁹ are as defined for the compound of formula (I).

In some embodiments, the compound is of formula (VI-A), (VI-B), (VI-C),or (VI-D).

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R^(5a),R^(5b), R⁶, R⁷, and R⁹ are as defined for the compound of formula (I).

TABLE 1 Representative Compounds of the Present Disclosure Compound No.Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

47

48

49

50

51

52

In some embodiments, provided is a compound selected from Compounds Nos.1-45 in Table 1, or a tautomer thereof, stereoisomer thereof, or apharmaceutically acceptable salt thereof. In some embodiments, providedis a compound selected from Compounds Nos. 47-52 in Table 1, or atautomer thereof, strereoisomer thereof, or a pharmaceuticallyacceptable salt thereof.

In one aspect, provided herein are methods of preparing the compounds ofFormula (I) described herein. In another aspect, provided herein areintermediate compounds for preparing the compounds of Formula (I). Alsoprovided herein are compounds which are assay probes, and which areoptionally tagged with, for example, a fluorescent label. In a furtheraspect, provided herein is a method for assaying inhibition of Cbl-b. Inone variation, provided herein is a method for assaying inhibition ofCbl-b comprising pre-incubating Cbl-b with an assay probe, such as anassay probe tagged with a fluorescent label, followed by exposing theCbl-b/assay probe mixture to a candidate compound, and then determiningwhether and to what extent the assay probe is displaced by the candidatecompound using, for example, FRET signal detection.

In any of the pharmaceutical compositions, methods, or kits describedherein, the Cbl-b inhibitor can be selected from one or more Cbl-binhibitors disclosed in the following patent application: compounds1-719 (including “a” and “b” variants thereof) of International PatentAppl. WO 2019/148005 or a compound of any of Formula (I-A), Formula (I),Formula (II-A), Formula (II), Formula (III-A), Formula (III), or Formula(IV) therein. The contents of International Patent Appl. WO 2019/148005is incorporated by reference herein in its entirety.

Representative compounds for use in the pharmaceutical compositions andmethods described herein are listed in Table 1A.

TABLE 1A Example No. Structure 53

54

55

56

57

58

59

60

61

In some embodiments, provided for use in the compositions and methodsdescribed herein is a compound selected from Compound Nos. 53-61 inTable 1A, or a tautomer thereof, stereoisomer thereof, or apharmaceutically acceptable salt thereof.

In one embodiment, the small molecule Cbl-b inhibitors for the methods,pharmaceutical compositions, and kits disclosed herein have a molecularweight of 1,000 daltons or less, or about 1,000 daltons or less. Inanother embodiment, the small molecule Cbl-b inhibitors have a molecularweight of 900 daltons or less, or about 900 daltons or less. In anotherembodiment, the small molecule Cbl-b inhibitors have a molecular weightof 800 daltons or less, or about 800 daltons or less. In anotherembodiment, the small molecule Cbl-b inhibitors have a molecular weightof 750 daltons or less, or about 750 daltons or less. In anotherembodiment, the small molecule Cbl-b inhibitors have a molecular weightof 700 daltons or less, or about 700 daltons or less. In anotherembodiment, the small molecule Cbl-b inhibitors have a molecular weightof 650 daltons or less, or about 650 daltons or less. In anotherembodiment, the small molecule Cbl-b inhibitors have a molecular weightof 600 daltons or less, or about 600 daltons or less. For any of theforegoing embodiments, the lower limit for the molecular weight of thesmall molecule Cbl-b inhibitors can be 100 daltons, 200 daltons, or 300daltons, or about 100 daltons, about 200 daltons, or about 300 daltons.

The schemes below describe methods of synthesizing the compoundsdisclosed herein. Mixtures of stereoisomers produced during synthesis,such as racemic mixtures of final compounds, can be separated into therespective enantiomers using common chromatography methods such assupercritical fluid chromatography in combination with chiral stationaryphases, chiral column chromatography, or other methods known in the art.

wherein Z¹, R⁶, and R¹⁰ are as defined for the compound of formula (I);X′ is Br or NH₂; R^(a) and R^(b) are suitable protecting groups; and(R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b),and R⁴ as described for the compound of formula (I).

Compounds of the general formula I-9 can be synthesized as outlined inScheme I. Coupling of boronic esters I-1 with alkenes I-2 under rhodiumcatalysis affords esters I-3. A triazole is assembled by hydrazideformation (I-4), cyclization and desulphurization, to provide compoundsI-6. Compounds I-6, wherein X′ is NH₂, can react with bromoesters I-8 toprovide compounds I-9, which are subsequently converted to compoundsI-10 by reductive amination with a substituted piperidine. CompoundsI-6, wherein X′ is Br, can react with piperidine derivatives I-7 underpalladium catalysis to directly give compounds I-10.

wherein Z¹, R⁶, R⁹, and R⁰ are as defined for the compound of formula(I); X′ is Br or NH₂; R^(a) and R^(b) are suitable protecting groups;and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b) and R⁴ as described for the compound of formula (I).

Compounds of the general formula I-9a can be synthesized as outlined inScheme I-a. Coupling of boronic esters I-1 with alkenes I-2 underrhodium catalysis affords esters I-3. A triazole is assembled byhydrazide formation (I-4), cyclization and desulphurization, to providecompounds I-6. Compounds I-6, wherein X′ is NH₂, can react withbromoesters I-8a to provide compounds I-9a, which are subsequentlyconverted to compounds I-10a by reductive amination with a substitutedpiperidine. Compounds I-6, wherein X′ is Br, can react with piperidinederivatives I-7a under palladium catalysis to directly give compoundsI-10a.

wherein R⁶ is as defined for the compound of formula (I), and (R)_(n)corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), and R⁴ asdescribed for the compound of formula (I).

Scheme II outlines a synthesis of compounds of the general formula II-4.Aldehydes II-1 are converted to piperidine derivatives II-2 by reductiveamination with a substituted piperidine, followed by ester hydrolysisunder basic conditions. Piperidines II-2 are then coupled to amines I-6with a coupling reagent such as HATU or T3P to afford compounds II-4.

wherein Z², R⁶, and R¹⁰ are as defined for the compound of formula (I);(R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b),and R⁴ as described for the compound of formula (I); R^(b) is a suitableprotecting group; Y^(b) is an alkyl group (such as methyl), a cycloalkylgroup (such as cyclopropyl), a haloalkyl group (such as bromomethyl) orCHO; and Y^(c) is hydroxyl or NH₂.

Scheme II-a outlines a synthesis of compounds of the general formulaII-5a. Compounds of formula II-1a can be converted to compounds II-2a byoxidation to an aldehyde with a reagent such as SeO₂, or brominated withBr₂, followed by reductive amination or direct displacement with asubstituted piperidine to form compounds of formula II-3a. CompoundsII-3a are then subjected to ester hydrolysis under basic conditions toafford compounds II-4a (Y^(c)=OH). Acids II-4a are then coupled toamines I-6 with a coupling reagent such as HATU or T3P to afford amidesII-5a.

wherein Z¹, R⁶, and R¹⁰ are as defined for the compound of formula (I),and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3n), and R⁴ as described for the compound of formula (I).

Compounds of the general formula III-4 can be prepared according toScheme III. Alcohol III-1 is converted to ester III-2 by conversion to ahalide, displacement with cyanide, then hydrolysis. The triazole isformed as in Scheme I. The bromide is converted to aniline III-3 bypalladium catalyzed coupling with t-butyl carbamate, followed bydeprotection. Compounds III-3 are then elaborated as in Scheme I toprovide compounds III-4.

wherein Z¹, R⁶, R⁹, and R¹⁰ are as defined for the compound of formula(I), and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

Compounds of the general formula III-4a can be prepared according toScheme III-a. Alcohol III-1 is converted to ester III-2 by conversion toa halide, displacement with cyanide, then hydrolysis. The triazole isformed as in Scheme I-a. The bromide is converted to aniline III-3 bypalladium catalyzed coupling with t-butyl carbamate, followed bydeprotection. Compounds III-3 are then elaborated as in Scheme I-a toprovide compounds III-4a.

wherein Z¹, R⁶, and R¹⁰ are as defined for the compound of formula (I),and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

Scheme IV shows an approach to preparing compounds of the generalformula IV-5. Ester IV-1 is converted to α-hydroxyester IV-2 bydeprotonation and treatment with an oxidizing agent such as anoxaziridine. The ester is elaborated to triazole IV-3 as in Scheme I.The hydroxy is converted to fluoride IV-4 by DAST. Compounds IV-4 arethen completed as in Scheme I to afford compounds IV-5.

wherein Z¹, R⁶, R⁹, and R⁰ are as defined for the compound of formula(I), and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R², R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

Scheme IV-a shows an approach to preparing compounds of the generalformula IV-5a. Ester IV-1 is converted to α-hydroxyester IV-2 bydeprotonation and treatment with an oxidizing agent such as anoxaziridine. The ester is elaborated to triazole IV-3 as in Scheme I-a.The hydroxy is converted to fluoride IV-4 by DAST. Compounds IV-4 arethen completed as in Scheme I-a to afford compounds IV-5a.

wherein R⁶, R⁷, R⁰, and Z¹ are as defined for the compound of formula(I), and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

A synthesis of compounds of general formula V-7 is provided in Scheme V.Trichloropyridine V-1 is coupled with dimethyl malonate to providediester V-2. Reaction of diester V-2 with allyl bromide, followed byreduction yields dihydroxide V-3. Compound V-3 is then reacted underMitsunobu conditions to afford oxetane V-4. The allyl group is oxidizedto an acid which is then converted to triazole V-5 as in Scheme I.Subsequent reaction of V-5 with nucleophiles such as amines or alcohols,or organometallic reagents under palladium catalysis yields V-6, whichis then coupled with indolones I-7 to provide compounds V-7.

wherein R⁶, R⁷, R⁹, R¹⁰, and Z¹ are as defined for the compound offormula (I), and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R²,R^(3a), R^(3b), and R⁴ as described for the compound of formula (I).

A synthesis of compounds of general formula V-7a is provided in SchemeV-a. Trichloropyridine V-1 is coupled with dimethyl malonate to providediester V-2. Reaction of diester V-2 with allyl bromide, followed byreduction yields dihydroxide V-3. Compound V-3 is then reacted underMitsunobu conditions to afford oxtetane V-4. The allyl group is oxidizedto an acid which is then converted to triazole V-5 as in Scheme I-a.Subsequent reaction of V-5 with nucleophiles such as amines or alcohols,or organometallic reagents under palladium catalysis yields V-6, whichis then coupled with indolones I-7a to provide compounds V-7a.

wherein R¹⁰ and Z¹ are as defined for the compound of formula (I), and(R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b),and R⁴ as described for the compound of formula (I).

Intermediate compounds of general formula I-7, employed in the generalsynthesis outlined in Scheme I, can be synthesized according to SchemeVI. Aldehydes VI-1 are coupled with substituted piperidines underreductive amination conditions to provide piperidine intermediates VI-2,which are treated with ammonia to provide compounds of formula I-7.

wherein R⁹, R¹⁰, and Z¹ are as defined for the compound of formula (I),and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

Intermediate compounds of general formula I-7a, employed in the generalsynthesis outlined in Scheme I-a, can be synthesized according to SchemeVI-a. Aldehydes VI-1a are coupled with substituted piperidines underreductive amination conditions to provide piperidine intermediatesVI-2a, which are treated with ammonia to provide compounds of formulaI-7a.

wherein R¹⁰ and Z¹ are as defined for the compound of formula (I), and(R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b),and R⁴ as described for the compound of formula (I).

Scheme VII outlines a synthesis for compounds of the general formulaVII-5. Bromide VII-1 is coupled with 3-nitrophenylboronic acid toprovide nitro VII-2. The ester of compound VII-2 is converted to atriazole as in Scheme I, and reduction of the nitro provides amineVII-3, which is then elaborated as in Scheme I to afford VII-5.

wherein R⁹, R¹⁰, and Z¹ are as defined for the compound of formula (I),and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I).

Scheme VII-a outlines a synthesis for compounds of the general formulaVII-5a. Bromide VII-1 is coupled with 3-nitrophenylboronic acid toprovide nitro VII-2. The ester of compound VII-2 is converted to atriazole as in Scheme I-a, and reduction of the nitro provides amineVII-3, which is then elaborated as in Scheme I-a to afford VII-5a.

wherein R⁹, R⁰, and Z¹ are as defined for the compound of formula (I),and (R)_(n) corresponds to R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), and R⁴ as described for the compound of formula (I), and P is aprotecting group such as pivaloyl.

Scheme VIII outlines a synthesis for compounds of the general formulaVIII-6. Hydroxy bromide VIII-1 is converted to a sulfide (VIII-2) bydisplacement with a thiol source such as tritylSH (Croft, Rosemary A. etal. Chemistry—A European Journal, 24(4), 818-821; 2018). The triazole isinstalled by S_(N)Ar displacement with a triazoyl chloride to affordVIII-4, which is coupled with an isoindolone such as I-5 to affordVIII-5. The protecting group is then removed under the action of Nicatalysis (Correa, Arkaitz et al., Journal of the American ChemicalSociety, 136(3), 1062-1069; 2014) to provide VIII-6 analogs.

Compounds 1, 2, 10, 22, 23, 24, 32, 36, 39, 40, 44, and 49-51 displayIC₅₀ values of less than 1 nM in the Cbl-b inhibition assay ofBiological Example 1, and in one embodiment are used for thepharmaceutical compositions and in the methods as disclosed herein.Compounds 3, 4, 11, 13-20, 25, 26, 28, 31, 33-35, 37, 38, 41-43, 45, 48,and 52 display IC₅₀ values of 1 nM-5 nM in the Cbl-b inhibition assay ofBiological Example 1, and in one embodiment are used for thepharmaceutical compositions and in the methods as disclosed herein.Compounds 5-9, 12, 21, 27, 29, 30, and 47 display IC₅₀ values of 5.1-100nM in the Cbl-b inhibition assay of Biological Example 1, and in oneembodiment are used for the pharmaceutical compositions and in themethods as disclosed herein.

In various embodiments, and as further described herein, compounds asprovided herein (as well as compositions comprising compounds describedherein, and methods using the compounds or compositions) have IC₅₀values of less than 1 nM, between 1 nM-5 nM, between 5.1-100 nM, orgreater than 100 nM, as determined by the Cbl-b inhibition assay ofBiological Example 1. In a further embodiment, and as further describedherein, compounds as provided herein (as well as compositions comprisingcompounds described herein, and methods using the compounds orcompositions) have IC₅₀ values of less than 1 nM, as determined by theCbl-b inhibition assay of Biological Example 1. In a further embodiment,and as further described herein, compounds as provided herein (as wellas compositions comprising compounds described herein, and methods usingthe compounds or compositions) have IC₅₀ values of between 1 nM-5 nM, asdetermined by the Cbl-b inhibition assay of Biological Example 1. In afurther embodiment, and as further described herein, compounds asprovided herein (as well as compositions comprising compounds describedherein, and methods using the compounds or compositions) have IC₅₀values of between 5.1-100 nM, as determined by the Cbl-b inhibitionassay of Biological Example 1. In a further embodiment, and as furtherdescribed herein, compounds as provided herein (as well as compositionscomprising compounds described herein, and methods using the compoundsor compositions) have IC₅₀ values greater than 100 nM, as determined bythe Cbl-b inhibition assay of Biological Example 1.

For IL-2 secretion from an immune cell (e.g., T-cell) co-stimulated withan anti-CD3 antibody and an anti-CD28 antibody, compounds as providedherein (as well as compositions comprising compounds described herein)induce ≤10 fold, between 11-15 fold, or greater than 15 fold change overbaseline, at inhibitor concentrations of 1 micromolar or 0.3 micromolar.

For IL-2 secretion from an immune cell (e.g., T-cell) stimulated with ananti-CD3 antibody, compounds as provided herein (as well as compositionscomprising compounds described herein) induce ≤0.33 fold, between0.34-0.66 fold, or greater than 0.66 fold change over baseline, atinhibitor concentrations of 3 micromolar or 1 micromolar.

For CD25 staining on the cell surface of an immune cell (e.g., T-cell)co-stimulated with an anti-CD3 antibody and an anti-CD28 antibody,compounds as provided herein (as well as compositions comprisingcompounds described herein) induce ≤1.24 fold, between 1.25-1.39 fold,or greater than 1.39 fold change over baseline, at inhibitorconcentrations 1 micromolar or 0.3 micromolar.

For CD25 staining on the cell surface of an immune cell (e.g., T-cell)stimulated with an anti-CD3 antibody, compounds as provided herein (aswell as compositions comprising compounds described herein) induce ≤1.04fold, between 1.05-1.14 fold, or greater than 1.14 fold change overbaseline, at inhibitor concentrations of 3 micromolar or 1 micromolar.

III. METHODS, MEDICAMENTS, AND USES

Provided herein are methods for modulating activity of an immune cell(e.g., a T-cell, a B-cell, or a NK-cell) such as by contacting theimmune cell with an effective amount of a Cbl-b inhibitor describedherein or a composition thereof. Also provided are in vitro methods ofproducing said immune cells with modulated activity, referred to hereinas “modified immune cells,” wherein said modified immune cells can beadministered to an individual in need thereof (e.g., an individualhaving cancer) by ex vivo methods. Further provided are in vivo methodsof modulating a response in an individual in need thereof (e.g., anindividual with cancer), wherein the method comprises administration ofan effective amount of a Cbl-b inhibitor described herein or acomposition thereof. Moreover, the present disclosure provides in vitromethods of producing an expanded population of lymphocytes after in vivolympho-conditioning in an individual, wherein the lympho-conditioningoccurs as a result of administration of an effective amount of a Cbl-binhibitor described herein or a composition thereof to the individual.In addition, the expanded population of lymphocytes can then beadministered to the individual with cancer. In some embodiments, themodified immune cells or the expanded population of lymphocytes areproduced from a biological sample comprising immune cells obtained fromthe individual, such as a blood sample comprising peripheral bloodmononuclear cells or a tumor biopsy comprising tumor infiltratinglymphocytes (TILs).

Additionally, provided are Cbl-b inhibitors for use as therapeuticactive substances. A Cbl-b inhibitor for use in treating or preventing adisease or condition associated with Cbl-b activity is provided. Also, aCbl-b inhibitor for use in treating cancer is provided. Further providedis the use of a Cbl-b inhibitor in the manufacture of a medicament fortreating or preventing a disease or condition associated with Cbl-bactivity. Also provided is the use of a Cbl-b inhibitor in themanufacture of a medicament for treating cancer. Moreover, the presentdisclosure provides treatment methods, medicaments, and uses comprisinga Cbl-b inhibitor as part of a combination therapy for treating cancerinvolving one or more of an immune checkpoint inhibitor, anantineoplastic agent, and radiation therapy.

In some embodiments of the treatment methods, medicaments, and uses ofthe present disclosure, the cancer is a hematologic cancer such aslymphoma, a leukemia, or a myeloma. In other embodiments of thetreatment methods, medicaments, and uses of the present disclosure, thecancer is a non-hematologic cancer such as a sarcoma, a carcinoma, or amelanoma.

Hematologic cancers include, but are not limited to, one or moreleukemias such as B-cell acute lymphoid leukemia (“BALL”), T-cell acutelymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL); one or morechronic leukemias including, but not limited to, chronic myelogenousleukemia (CML) and chronic lymphocytic leukemia (CLL); additionalhematologic cancers or hematologic conditions including, but not limitedto, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cellneoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicularlymphoma, hairy cell leukemia, small cell- or a large cell-follicularlymphoma, malignant lymphoproliferative conditions, MALT lymphoma,mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma,myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, and “preleukemia,” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells.

Non-hematologic cancers include but are not limited to, a neuroblastoma,renal cell carcinoma, colon cancer, colorectal cancer, breast cancer,epithelial squamous cell cancer, melanoma, stomach cancer, brain cancer,lung cancer (e.g., NSCLC), pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, prostate cancer, testicularcancer, thyroid cancer, uterine cancer, adrenal cancer, and head andneck cancer.

In some aspects, the effectiveness of administration of a Cbl-binhibitor in the treatment of a disease or disorder such as cancer ismeasured by assessing clinical outcome, such as reduction in tumor sizeor number of tumors, and/or survival. In some embodiments, “treatingcancer” comprises assessing a patient's response to the treatmentregimen according to the Response Evaluation Criteria in Solid Tumors(RECIST version 1.1) as described (see, e.g., Eisenhauer et al., Eur JCancer, 45:228-247, 2009; and Nishino et al., Am J Roentgenol, 195:281-289, 2010). Response criteria to determine objective anti-tumorresponses per RECIST 1.1 include: complete response (CR); partialresponse (PR); progressive disease (PD); and stable disease (SD).

A. Isolation and Processing of Cells

Provided are methods for the preparation and processing of immune cellsproduced (e.g., modified immune cells) and used in the methods herein.As used herein, the term “modified immune cells” refers to immune cellsor a cell population comprising the immune cells which have beencultured, incubated, and/or have been contacted with an effective amountof a Cbl-b inhibitor to modulate the activity of said immune cells. Insome embodiments, the modified immune cells can be used forimmunotherapy, such as in connection with adoptive immunotherapymethods.

1. Samples

In some embodiments, the immune cells to be modified or cell populationscomprising the immune cells to be modified are isolated from a sample,such as a biological sample, e.g., one obtained from or derived from anindividual (e.g., a human). In some embodiments, the individual fromwhich the immune cell is isolated is one having a particular disease orcondition (e.g., cancer) or in need of a cell therapy or to which celltherapy will be administered. The individual in some embodiments is ahuman in need of a particular therapeutic intervention, such as theadoptive cell therapy for which immune cells are being isolated,processed, and/or modified. Accordingly, the cells isolated from theindividual in some embodiments are primary cells (e.g., primary humancells). As used herein, the term “primary cells” refers to cellsisolated directly from mammalian biological fluid or tissue (e.g., humanbiological fluid or tissue).

In some embodiments, the immune cells to be modified are hematopoieticcells, multipotent stem cells, myeloid progenitor cells, lymphoidprogenitor cells, T-cells, B-cells, and/or NK-cells. As used herein, theterm “hematopoietic cells” includes hematopoietic stem cells andhematopoietic progenitor cells. In some embodiments, the immune cells tobe modified are present in a heterogeneous cell population or acomposition comprising a heterogeneous cell population. For example, theimmune cells to be modified may be hematopoietic cells present in aheterogeneous cell population comprising cells such as differentiatedcells derived from a tissue or organ. In some embodiments, the immunecells to be modified are present in a homogenous cell population or acomposition comprising a homogenous cell population. For example, theimmune cells to be modified may be hematopoietic cells present in ahomogenous cell population comprising only hematopoietic cells. In someembodiments, the immune cells to be modified or cell populationscomprising the immune cells to be modified include one or more subsetsof immune cells. For example, one or more subsets of immune cells may beCD4+ cells, CD8+ cells and subpopulations thereof, such as those definedby function, activation state, maturity, potential for differentiation,expansion, localization, persistence capacities, surface marker profile,cytokine secretion profile, and/or degree of differentiation.

In some embodiments, biological samples described herein include tissue,fluid, and other samples taken directly from the individual, as well assamples resulting from one or more processing steps, such as separation,centrifugation, genetic engineering (e.g., transduction with a viralvector encoding a recombinant chimeric receptor), washing, and/orincubation. The biological sample can be a sample obtained directly froma biological source or a sample that is processed. Biological samplesinclude, but are not limited to, body fluids, such as blood, plasma,serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue andorgan samples (e.g., sample from a tissue or organ containing a tumor),including processed samples derived therefrom. In some embodiments, thebiological sample is a biological fluid sample or a biological tissuesample. In some embodiments, the biological sample is a biologicaltissue sample. In some aspects, the biological sample from which theimmune cells are derived or isolated is blood or a blood-derived sample,or is derived from an apheresis or leukapheresis product.

Exemplary biological samples include whole blood, peripheral bloodmononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissuebiopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoidtissue, mucosa associated lymphoid tissue, spleen, other lymphoidtissues, liver, lung, stomach, intestine, colon, kidney, pancreas,breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ,and/or cells derived therefrom. Biological samples include, in thecontext of cell therapy (e.g., adoptive cell therapy) samples fromautologous sources (i.e., obtained from or derived from the individualin need of cell therapy) and allogeneic sources (i.e., obtained from orderived from an individual or source other than the individual in needof cell therapy).

In some embodiments, the immune cells to be modified or a cellpopulation comprising the immune cells to be modified are derived from acell line (e.g., a T-cell line, a B-cell line, a NK-cell line, etc.). Insome embodiments, the immune cells to be modified or a cell populationcomprising the immune cells to be modified are obtained from axenogeneic source, such as from mouse, rat, non-human primate, or pig.

2. Cell Processing and Separation

In some embodiments, isolation of the immune cells to be modifiedincludes one or more preparation and/or cell separation steps. The oneor more cell separation steps can be non-affinity based separation oraffinity based separation. As an example, non-affinity based separationcan be centrifugation of a composition comprising the immune cells to bemodified. In some embodiments, the non-affinity based separation methodsinclude density-based cell separation methods, such as the preparationof white blood cells from peripheral blood by lysing the red blood cellsand centrifugation through a Percoll or Ficoll gradient. Affinity-basedseparation methods can include contacting a composition comprising theimmune cells to be modified with antibody coated beads. Antibody-coatedbeads contemplated herein include, but are not limited to, magneticbeads (e.g., Dynabeads® marketed by Life Technologies, Carlsbad, Calif.,MACS® microbeads marketed by Miltenyi Biotec Inc., Auburn, Calif.; orEasySep™ Direct RapidSpheres™ marketed by Stemcell Technologies,Vancouver, BC, Canada) coated with an antibody that binds to a markerexpressed on the surface of the immune cell to be modified. In someembodiments, specific subpopulations of T-cells, such as cells positivefor or otherwise expressing high levels of one or more surface markers,e.g., CD4+, CD8+, etc., are isolated by positive or negative selectiontechniques. Positive selection can be based on a technique in which thetarget cells (e.g., immune cells to be modified) have bound to a reagentand are retained for further use. For example, T-cells that are CD3+ canbe positively selected using magnetic beads conjugated to anti-CD3antibodies (e.g., MACS® CD3 human microbeads). Negative selection can bebased on a technique in which the targets cells (e.g., immune cells tobe modified) that have not bound to a reagent are retained. For example,total human primary T-cells can be isolated from peripheral bloodmononuclear cells (PMBCs) utilizing negative selection, wherein acocktail of antibodies against surface markers CD14, CD15, CD16, CD19,CD34, CD36, CD56, CD123, and CD235a are incubated in a sample comprisingthe PBMCs before passing the sample by magnetic beads for removal ofcells expressing those surface markers and retaining the remaining cellsin the sample for subsequent processing. In some embodiments, the immunecells or a cell population comprising the immune cells to be modifiedare washed, centrifuged, and/or incubated in the presence of one or morereagents, for example, to remove unwanted components, enrich for desiredcomponents, lyse or remove cells sensitive to particular reagents. Insome examples, the immune cells are separated based on one or moreproperty, such as density, adherent properties, size, sensitivity,and/or resistance to particular components. Cell separation steps do notrequire 100% enrichment or removal of particular cells. In someembodiments, positive selection of or enrichment for immune cells of aparticular type (e.g., CD4+ T-cells) refers to increasing the number orpercentage of such cells. In some embodiments, removal, or depletion ofcells of a particular type that are not of interest such as by negativeselection, refers to decreasing the number or percentage of such cells.

In some embodiments, immune cells or a cell population comprising theimmune cells are obtained from the circulating blood of an individual,e.g., by apheresis or leukapheresis. In some aspects, a samplecomprising the immune cells to be modified contains lymphocytes,including T-cells, B-cells, and NK-cells, as well as monocytes,granulocytes, red blood cells, and/or platelets, and in some aspectscontains cells other than red blood cells and platelets.

In some embodiments, the blood cells collected from the individual arewashed such as to remove the plasma fraction and to place the cellpopulation comprising the immune cells to be modified in an appropriatebuffer or media for subsequent processing steps. In some embodiments,the cell population comprising the immune cells to be modified is washedwith phosphate-buffered saline. In some embodiments, the wash solutionlacks calcium and/or magnesium. In some aspects, a washing step isaccomplished by a semi-automated “flow-through” centrifuge. In someaspects, a washing step is accomplished by tangential flow filtration.In some embodiments, the immune cells to be modified or cell populationcontaining the immune cells to be modified are resuspended in a varietyof suitable buffers after washing, such as, for example, calcium and/ormagnesium-free phosphate-buffered saline. In some embodiments,components of a blood cell sample are removed and the immune cells to bemodified or a cell population comprising the immune cells to be modifiedare directly resuspended in a suitable cell culture medium.

Representative methods for processing and/or separating immune cells,such as hematopoietic cells from samples containing a cell populationcontaining said hematopoietic cells (e.g., samples comprising PBMCs),are described in Biological Example 2 and Biological Example 3 herein.Methods and techniques for processing and/or separating immune cellssuch as hematopoietic cells, multipotent stem cells, myeloid progenitorcells, lymphoid progenitor cells, T-cells, B-cells, and/or NK-cells arewell known in the art. See for example, U.S. Patent Application No.2017/0037369; U.S. Patent Application No. 2012/0148553; U.S. Pat. Nos.6,461,645; 6,352,694; and 7,776,562.

3. Incubation and Treatment

Provided herein are methods for modulating the activity of an immunecell, such as the processed and/or separated immune cells describedabove, by contacting the immune cell with an effective amount of a Cbl-binhibitor described herein. Also provided herein are modified immunecells produced by any of the methods described herein such as byculturing a cell population containing an immune cell (e.g., theprocessed and/or separated immune cells described above) in the presenceof an effective amount of a Cbl-b inhibitor to modulate the activity ofthe immune cell and thereby produce the modified immune cell.

In some embodiments, the immune cells to be modified (e.g., theprocessed and/or separated immune cells described above) are incubatedand/or cultured in a suitable culture medium prior to contacting saidimmune cells with a Cbl-b inhibitor provided herein. In someembodiments, the immune cells to be modified are incubated and/orcultured in a suitable culture medium simultaneously to contacting saidimmune cells with a Cbl-b inhibitor provided herein.

The processed and/or separated immune cells to be modified or cellpopulation comprising the immune cells to be modified can bedifferentiated and/or expanded in vitro. In some embodiments, the immunecells to be modified are hematopoietic cells, multipotent stem cells,myeloid progenitor cells, lymphoid progenitor cells, T-cells, B-cells,and/or NK-cells. In some embodiments, the immune cell to be modified isincubated in a suitable cell culture medium comprising a Cbl-b inhibitordescribed herein before differentiation and/or expansion of the immunecell. In some embodiments, the immune cell to be modified is incubatedin a suitable cell culture medium comprising a Cbl-b inhibitor describedherein after differentiation and/or expansion of the immune cell. Theimmune cells become modified (i.e., modified immune cells) upon contactwith a Cbl-b inhibitor provided herein in an effective amount tomodulate the activity of said immune cells. In some embodiments, theimmune cell to be modified is not differentiated and/or expanded invitro and is therefore the same cell type as the modified immune cellthat has been contacted with a Cbl-b inhibitor. For example, a T-cellcan be incubated in a suitable medium comprising a Cbl-b inhibitorwithout differentiation of the T-cell. In other embodiments, the immunecell to be modified is differentiated and/or expanded in vitro and istherefore a different cell type than the modified immune cell that hasbeen contacted with a Cbl-b inhibitor. For example, a hematopoietic cellcan be incubated in a suitable medium comprising a Cbl-b inhibitor aswell as other agents that drive differentiation of the hematopoieticcell into a mature hematopoietic cell. Accordingly, in some aspects ofthe embodiments herein, the modified immune cells are hematopoieticcells, multipotent stem cells, myeloid progenitor cells, lymphoidprogenitor cells, T-cells, B-cells, and/or NK-cells. Methods forexpansion and/or differentiation of immune cells are well known in theart. See, for example, International Patent Application No. WO2017/037083.

An effective amount of a Cbl-b inhibitor is the amount or concentrationof the Cbl-b inhibitor that is sufficient to modulate the activity ofthe immune cell as compared to a reference sample. The reference samplemay be immune cells that have not been contacted with the Cbl-binhibitor. In some embodiments, the concentration of a Cbl-b inhibitoradded to a composition (e.g., cell culture medium) comprising the immunecells to be modified is from about 1 pM to about 100 μM, about 5 pM toabout 100 μM, about 10 pM to about 100 μM, about 20 pM to about 100 μM,about 40 pM to about 100 μM, about 60 pM to about 100 μM, about 80 pM toabout 100 μM, about 1 nM to about 100 μM, about 3 nM to about 100 μM,about 10 nM to about 100 μM, about 15 nM to about 100 μM, about 20 nM toabout 100 μM, about 40 nM to about 100 μM, about 60 nM to about 100 μM,about 80 nM to about 100 μM, about 0.1 μM to about 100 μM, about 0.1 μMto about 90 μM, about 0.1 μM to about 80 μM, about 0.1 μM to about 70μM, about 0.1 μM to about 60 μM, about 0.1 μM to about 50 μM, about 0.1μM to about 40 μM, about 0.1 μM to about 30 μM, about 0.1 μM to about 20μM, about 0.1 μM to about 10 μM, about 0.2 μM to about 10 μM, or about0.3 μM to about 8 μM. In some embodiments, the concentration of a Cbl-binhibitor added to a composition (e.g., cell culture medium) comprisingthe immune cells to be modified is about 1 pM, about 2 pM, about 3 pM,about 4 pM, about 5 pM, about 10 pM, about 20 pM, about 30 pM, about 40pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM,about 1 nM, about 3 nM, about 5 nM, about 10 nM, about 20 nM, about 40nM, about 50 nM, about 80 nM, about 0.1 μM, about 0.2 μM, about 0.3 μM,about 0.4 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about15 μM, about 20 μM, about 25 μM, about 30 μM, about 40 μM, about 50 μM,about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM. Insome embodiments, the concentration of a Cbl-b inhibitor added to acomposition (e.g., cell culture medium) comprising the immune cells tobe modified is about 0.3 μM, about 1 μM, or about 4 μM. In someembodiments, the concentration of a Cbl-b inhibitor added to acomposition (e.g., cell culture medium) comprising the immune cells tobe modified is about 1 μM or about 8 μM.

The effective amount of a Cbl-b inhibitor is in contact with the immunecells for a sufficient time to modulate the activity of the immune cellas compared to a reference sample. The reference sample may be immunecells that have not been contacted with the Cbl-b inhibitor but areincubated for the same length of time as the composition (e.g., cellculture medium) comprising the immune cells and the Cbl-b inhibitor. Insome embodiments, the Cbl-b inhibitor is in contact and/or is incubatedwith the immune cells from about 1 minute to about 1 hour, about 5minutes to about 1 hour, about 10 minutes to about 1 hour, about 15minutes to about 1 hour, about 20 minutes to about 1 hour, about 30minutes to about 1 hour, about 45 minutes to about 1 hour, about 1 hourto about 2 hours, about 1 hour to about 4 hours, about 1 hour to about 6hours, about 1 hour to about 8 hours, about 1 hour to about 12 hours,about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 6hours to about 7 hours, about 6 hours to about 24 hours, about 8 hoursto about 24 hours, about 10 hours to about 24 hours, about 15 hours toabout 24 hours, about 20 hours to about 24 hours, about 12 hours toabout 48 hours, about 24 hours to about 48 hours, or about 36 hours toabout 48 hours. In some embodiments, the Cbl-b inhibitor is in contactand/or is incubated with the immune cells for about 1 minute, about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours,about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22hours, or about 24 hours. In some embodiments, the Cbl-b inhibitor is incontact and/or is incubated with the immune cells from about 1 day toabout 7 days, about 2 days to about 7 days, about 3 days to about 7days, about 4 days to about 7 days, about 5 days to about 7 days, orabout 6 days to about 7 days. In some embodiments, the Cbl-b inhibitoris in contact and/or is incubated with the immune cells from about 7days to about 14 days, about 14 days to about 21 days, or about 21 daysto about 28 days. In some embodiments, the Cbl-b inhibitor is in contactand/or is incubated with the immune cells for about 1 day, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days, about 7 days,about 8 days, about 9 days, about 10 days, about 11 days, about 12 days,about 13 days, or about 14 days.

In some embodiments, the immune cells or a cell population comprisingthe immune cells are incubated under a suitable condition to induceproliferation, expansion, activation, and/or survival of the immunecells. Suitable conditions during incubation include, but are notlimited to, use of one or more of cell culture medium, temperature,incubation time, the presence of a stimulating agent (e.g., anti-CD3and/or anti-CD28 antibody), and the presence of any other beneficialagents, such as growth factors, cytokines, chemokines, and/orrecombinant soluble receptors.

In some embodiments, a suitable condition to induce proliferation,expansion, activation, and/or survival of the immune cells includes theprovision of stimulating conditions comprising agents that are capableof activating the immune cell (e.g., NK-cell). For example, a suitablecondition to induce proliferation, expansion, activation, and/orsurvival of a T-cell includes the provision of stimulating conditionscomprising agents that are capable of activating intracellular signalingin the T-cell. Full activation of T-cells generally requires therecognition of antigen by the T-cell receptor, referred to herein as“TCR” (signal one) as well as recognition of costimulators such as CD28(signal two). In some aspects, one or more agents turn on or initiate aTCR complex-mediated intracellular signaling cascade in a T-cell. Forexample, a first agent can bind to a component of the TCR complex inorder to activate the T-cell and a second agent can bind to acostimulatory molecule on the surface of the T-cell to thereby stimulatethe activated T-cell. In some embodiments, the first agent stimulated aTCR/CD3 complex-associated signal in the T-cell by specifically bindingto CD3 (e.g., an anti-CD3 antibody). In a further embodiment, theco-stimulatory molecule on the surface of the T-cell may be CD28 and thesecond agent specifically binds to CD28 (e.g., anti-CD28 antibody). Suchagents include, but are not limited to, antibodies, divalent antibodyfragments, and binding molecules such as those specific for a TCRcomplex component (e.g., anti-CD3 antibody) and/or those specific forcostimulatory receptor (e.g., anti-CD28 antibody). In some embodiments,an agent that specifically binds to CD3 is an anti-CD3 antibody, adivalent antibody fragment of an anti-CD3 antibody (e.g., (Fab)2′fragment or a divalent scFv fragment), a monovalent antibody fragment ofan anti-CD3 antibody (e.g., a Fab fragment, a Fv fragment, or a scFvfragment), or a CD3 binding molecule (e.g., an aptamer). In someembodiments, an agent that specifically binds to CD28 is an anti-CD28antibody, a divalent antibody fragment of an anti-CD28 antibody (e.g.,(Fab)2′ fragment or a divalent scFv fragment), a monovalent antibodyfragment of an anti-CD28 antibody (e.g., a Fab fragment, a Fv fragment,or a scFv fragment), and a CD28 binding molecule (e.g., an aptamer). Theone or more agents provided herein (e.g., anti-CD3 antibody andanti-CD28 antibody) for example, can be bound to a solid support such asa bead, or cross-linked with an anti-Fc antibody. In some embodiments,the expansion method step may further comprise the step of addinganti-CD3 antibody and/or anti-CD28 antibody to the culture medium. Insome embodiments, the stimulating agents added to the cell culturemedium include one or more cytokines such as, but not limited to one ormore of IL-2, IL-7, IL-15, and IL-21. For example, IL-2 can be added ata concentration of at least about 10 units/mL to a cell culture mediumcomprising the immune cells and agents such as anti-CD3 antibodiesand/or anti-CD28 antibodies.

In some embodiments, a suitable condition to induce proliferation,expansion, activation, and/or survival of a T-cell includes theprovision of stimulating conditions or agents which are capable ofactivating intracellular signaling through the T-cell receptor (TCR)complex, and a Cbl-b inhibitor as described herein. In some embodiments,the immune cells or a cell population comprising the immune cells areincubated with a first agent that stimulates a TCR/CD3complex-associated signal in the T-cell by specifically binding to CD3(e.g., an anti-CD3 antibody). In a further embodiment, the immune cellsor a cell population comprising the immune cells are incubated with afirst agent that stimulates a TCR/CD3 complex-associated signal in theT-cell by specifically binding to CD3 (e.g., an anti-CD3 antibody), witha second agent that binds to the co-stimulatory molecule CD28 (e.g., ananti-CD28 antibody), and with a Cbl-b inhibitor at a concentration ofabout 1 pM to about 100 μM (e.g., about 0.3 μM, about 1 μM, or about 4μM). In some embodiments, a suitable condition to induce proliferation,expansion, activation, and/or survival of a T-cell when in the presenceof a Cbl-b inhibitor does not require stimulation through aco-stimulatory molecule (e.g., CD28). Contacting T-cells with a Cbl-binhibitor or a composition thereof can bypass the need forco-stimulation required for T-cells to enter into an activated state. Incertain embodiments, the immune cells or a cell population comprisingthe immune cells are incubated with a first agent that stimulates aTCR/CD3 complex-associated signal in the T-cell by specifically bindingto CD3 (e.g., an anti-CD3 antibody) and with a Cbl-b inhibitor at aconcentration of about 0.001 μM to about 1,000 μM, about 0.01 μM toabout 100 μM, about 0.1 μM to about 10 μM, or about 0.1 μM to about 50μM (e.g., about 1 μM or about 8 μM).

In some embodiments of the methods for modulating activity of an immunecell, the immune cell is a T-cell and modulating activity of the T-cellcomprises increased T-cell activation and/or increased T-cellproliferation. T-cells contemplated in embodiments herein may be in atolerant state even in the presence of an activating agent that binds toa component of the TCR complex, such as an anti-CD3 antibody, as well asin the presence of a stimulating agent that binds a co-stimulatorymolecule, such as an anti-CD28 antibody. In some embodiments, the methodof modulating activity of a T-cell comprises contacting the T-cell withan effective amount of a Cbl-b inhibitor or a composition thereof in thepresence of an anti-CD3 antibody in combination with an anti-CD28antibody. In some embodiments, the method of modulating activity of aT-cell comprises contacting the T-cell with an effective amount of aCbl-b inhibitor or a composition thereof, wherein the T-cell previouslyhas been in contact with an anti-CD3 antibody in combination with ananti-CD28 antibody. In some embodiments, stimulation via theco-stimulatory CD28 molecule is not required for modulating the activityof the T-cell (e.g., increasing T-cell activation and/or increasingT-cell proliferation). In some embodiments, the method of modulatingactivity of a T-cell comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof in the presence ofan anti-CD3 antibody alone. In some embodiments, the method ofmodulating activity of a T-cell comprises contacting the T-cell with aneffective amount of a Cbl-b inhibitor or a composition thereof, whereinthe T-cell has previously been in contact with one or more agents thatactivate the T-cell (e.g., an anti-CD3 antibody), wherein said agents donot include an agent that stimulates the CD28 co-stimulatory molecule(e.g., an anti-CD28 antibody).

In some embodiments, the immune cell is a T-cell, and modulatingactivity of the T-cell comprises enhanced T-cell activation and/orenhanced T-cell proliferation. For example, T-cells contemplated inembodiments herein may be in an activated state such as when in thepresence of agents that activate the T-cells (e.g., anti-CD3 antibody),and in some further embodiments, in the presence of agents thatstimulate the T-cells (e.g., anti-CD28 antibody). Contacting T-cellswith a Cbl-b inhibitor or composition thereof can lower the thresholdrequired for activation and therefore enhance activation and/orproliferation of T-cells that are in the presence of an activating agent(e.g., an anti-CD3 antibody) and in some further embodiments, astimulating agent (e.g., an anti-CD28 antibody). In some embodiments,the method of modulating activity of a T-cell comprises contacting theT-cell with an effective amount of a Cbl-b inhibitor or a compositionthereof in the presence of an anti-CD3 antibody in combination with ananti-CD28 antibody. In some embodiments, the method of modulatingactivity of a T-cell comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof, wherein the T-cellhas previously been in contact with an anti-CD3 antibody in combinationwith an anti-CD28 antibody. In some embodiments, stimulation via theco-stimulatory CD28 molecule is not required for modulating the activityof the T-cell (e.g., enhancing T-cell activation and/or enhancing T-cellproliferation). In some embodiments, the method of modulating activityof a T-cell comprises contacting the T-cell with an effective amount ofa Cbl-b inhibitor or a composition thereof in the presence of ananti-CD3 antibody alone. In some embodiments, the method of modulatingactivity of a T-cell comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof, wherein the T-cellhas previously been in contact with one or more agents that activate theT-cell (e.g., anti-CD3 antibody).

In some embodiments, the immune cell is a T-cell and modulating activityof the T-cell comprises decreased T-cell dysfunction including decreasedT-cell exhaustion, decreased T-cell tolerance, and/or decreased T-cellanergy. General principles of T-cell dysfunction are well known in theart (see e.g., Schietinger et al., Trends Immunol., 35: 51-60, 2014).Immune tolerance is a process that is part of the normal function of theimmune system. Antigen-specific immune tolerance is characterized by adecrease in responsiveness to an antigen, which is induced by previousexposure to that antigen. When specific lymphocytes (e.g., T-cells)encounter antigens, the lymphocytes may be activated, leading to anantigen-specific immune response, or the lymphocytes (e.g., T-cells) maybe inactivated or eliminated, leading instead to antigen-specific immunetolerance. In some aspects, tolerance can be caused by clonal anergy,peripheral clonal deletion, suppression of T-cells, and/or other formsof antigen-specific tolerance. In some embodiments, tolerance may resultfrom or be characterized by the induction of anergy. In some aspects,anergy can result from exposure of T-cells to an antigen in the absenceof costimulation. Prolonged antigen recognition by the TCR alone, in theabsence of the co-stimulatory signal, may lead to anergy (i.e.,functional unresponsiveness). Anergic T-cells may be refractory tosubsequent antigenic challenge and may be capable of suppressing otherimmune responses. Generally, in the natural setting, tolerance isinvolved in non-reactivity or nonproductive reactivity to self-antigens.In some cases, however, tolerance to a “non-self” antigen can beinduced. Thus, in some aspects, the same mechanisms by which matureT-cells that recognize self-antigens in peripheral tissues becomeincapable of subsequently responding to these antigens also may regulateunresponsiveness to foreign or “non-self” antigens such as thoseexpressed by cancer cells. Accordingly, T-cells contemplated inembodiments herein may be in a tolerant state even in the presence ofstimulatory agents such as agents that bind to a co-stimulatory moleculesuch as CD28. Contacting T-cells with a Cbl-b inhibitor provided hereinor a composition thereof can bypass aspects of T-cell dysfunction suchas T-cell tolerance, T-cell anergy, and/or T-cell exhaustion. In someembodiments, the method of modulating activity of a T-cell (e.g.,decreasing T-cell tolerance, decreasing T-cell anergy, and/or decreasingT-cell exhaustion) comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof. In someembodiments of the methods herein, modulating activity of a T-cell(e.g., decreasing T-cell tolerance, decreasing T-cell anergy, and/ordecreasing T-cell exhaustion) comprises contacting the T-cell with aneffective amount of a Cbl-b inhibitor or a composition thereof in thepresence of an anti-CD3 antibody in combination with an anti-CD28antibody. In some embodiments of the methods herein, the method ofmodulating activity of a T-cell (e.g., decreasing T-cell tolerance,decreasing T-cell anergy, and/or decreasing T-cell exhaustion) comprisescontacting the T-cell with an effective amount of a Cbl-b inhibitor or acomposition thereof, wherein the T-cell previously has been in contactwith an anti-CD3 antibody in combination with an anti-CD28 antibody. Insome embodiments, stimulation via the co-stimulatory CD28 molecule isnot required for modulating the activity of the T-cell (e.g., decreasingT-cell tolerance, decreasing T-cell anergy, and/or decreasing T-cellexhaustion). In some embodiments of the methods herein, the method ofmodulating activity of a T-cell (e.g., decreasing T-cell tolerance,decreasing T-cell anergy, and/or decreasing T-cell exhaustion) comprisescontacting the T-cell with an effective amount of a Cbl-b inhibitor or acomposition thereof in the presence of an anti-CD3 antibody alone. Insome embodiments, the method of modulating activity of a T-cell (e.g.,decreasing T-cell tolerance, decreasing T-cell anergy, and/or decreasingT-cell exhaustion) comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof, wherein the T-cellhas previously been in contact with one or more agents that activate theT-cell, such as an anti-CD3 antibody alone.

T-cell activation and T-cell tolerance are tightly controlled processesregulating the immune response. Accordingly, provided herein are methodsof modulating activity of the T-cell, wherein modulating activity of theT-cell comprises increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and/or decreased T-celltolerance. In some embodiments, the method of modulating activity of aT-cell (e.g., increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and/or decreased T-celltolerance) comprises contacting the T-cell with an effective amount of aCbl-b inhibitor or a composition thereof. In some embodiments of themethods herein, modulating activity of a T-cell (e.g., increasing T-cellactivation, increasing T-cell proliferation, decreasing T-cellexhaustion, and/or decreasing T-cell tolerance) comprises contacting theT-cell with an effective amount of a Cbl-b inhibitor or a compositionthereof in the presence of an anti-CD3 antibody in combination with ananti-CD28 antibody. In some embodiments of the methods herein, themethod of modulating activity of a T-cell (e.g., increasing T-cellactivation, increasing T-cell proliferation, decreasing T-cellexhaustion, and/or decreasing T-cell tolerance) comprises contacting theT-cell with an effective amount of a Cbl-b inhibitor or a compositionthereof, wherein the T-cell previously has been in contact with ananti-CD3 antibody in combination with an anti-CD28 antibody. In someembodiments, stimulation via the co-stimulatory CD28 molecule is notrequired for modulating the activity of the T-cell (e.g., increasingT-cell activation, increasing T-cell proliferation, decreasing T-cellexhaustion, and/or decreasing T-cell tolerance). In some embodiments ofthe methods herein, the method of modulating activity of a T-cell (e.g.,increasing T-cell activation, increasing T-cell proliferation,decreasing T-cell exhaustion, and/or decreasing T-cell tolerance)comprises contacting the T-cell with an effective amount of a Cbl-binhibitor provided herein or a composition thereof in the presence of ananti-CD3 antibody alone. In some embodiments, the method of modulatingactivity of a T-cell (e.g., increasing T-cell activation, increasingT-cell proliferation, decreasing T-cell exhaustion, and/or decreasingT-cell tolerance) comprises contacting the T-cell with an effectiveamount of a Cbl-b inhibitor or a composition thereof, wherein the T-cellhas previously been in contact with one or more agents that activate theT-cell (e.g., an anti-CD3 antibody).

In some embodiments of the methods herein, increased T-cell activationcomprises increased production of one or more cytokines from T-cells orsurrounding immune cells in the activated T-cell microenvironment (e.g.,myeloid cells). In some embodiments, the one or more cytokines include,but are not limited to: IFN-7, IL-10, IL-2, IL-4, IL-5, IL-6, IL-13,IL-18, TNFα, and GM-CSF. In some embodiments, the cytokine is one ormore of: IL-2, IFN-7, TNFα, and GM-CSF. In some embodiments, thecytokine is a chemokine. In some embodiments, the one or more chemokinesinclude, but are not limited to: IP-10, Eotaxin, GRO alpha, RANTES,MIP-1α, MIP-1β, MIP-2, MCP-1, and MCP-3. Increased expression ofcytokines can be measured by ELISA.

In some embodiments of the methods herein, increased T-cell activationcomprises increased cell surface expression of one or more T-cellactivation markers. In some embodiments, the one or more T-cellactivation markers include, but are not limited to: CD25, CD44, CD62L,CD69, CD152 (CTLA4), CD154, CD137, and CD279. In some embodiments, theT-cell activation marker is one or more of: CD25, CD69, and CTLA4.Increased expression of cell surface markers can be measured by FACS.

Methods for experimentally determining increased T-cell activation,increased T-cell proliferation, decreased T-cell exhaustion, and/ordecreased T-cell tolerance are well known in the art. In someembodiments, representative methods of determining T-cell activation canbe found in Biological Example 2 provided herein. In some embodiments,representative in vitro and in vivo methods of determining increasedT-cell activation, increased T-cell proliferation, decreased T-cellexhaustion, and/or decreased T-cell tolerance can be found in BiologicalExample 3 provided herein.

In some embodiments of the methods for modulating activity of an immunecell, the immune cell is a B-cell and modulating activity of the B-cellcomprises increased B-cell activation. In some embodiments, increasedB-cell activation comprises increased cell surface expression of one ormore B-cell activation markers. In some embodiments, the one or moreB-cell activation markers include, but are not limited to: CD69, CD86,and MHC class II (e.g., HLA-DR). In some embodiments, the B-cellactivation marker is CD69. Increased expression of cell surface markerscan be measured by FACS. In some embodiments, increased B-cellactivation comprises increased activation of proteins in signalingpathways such as those mediated by ERK, INK, and Syk. Increasedactivation of said proteins can be detected by measurement of levels ofphosphorylation on the proteins using reagents such as anti-phosphoantibodies available in the art.

In some embodiments of the methods for modulating activity of an immunecell, the immune cell is a NK-cell and modulating activity of theNK-cell comprises increased NK-cell activation. In some embodiments,increased NK-cell activation comprises secretion of one or morecytokines. In some embodiments, the one or more cytokines include, butare not limited to: IFN-γ, TNFα, and MIP-1β. Increased expression ofcytokines can be measured by ELISA. In some embodiments, increasedNK-cell activation comprises increased cell surface expression of one ormore NK-cell activation markers. In some embodiments, the one or moreNK-cell activation markers include, but are not limited to: CD69, andCD107a. Increased expression of cell surface markers can be measured byFACS. In some embodiments, increased NK-cell activation comprisesincreased killing of target cells such as tumor cells, including primarytumor cells, and cell line derived tumor cells such as the K562 cellline.

Methods for experimentally determining increased B-cell activation andNK-cell activation are well known in the art (see e.g., Fauriat et al.,Blood. 115: 2167-76, 2010; Beano et al., J. Transl. Med., 6:25 2008;Claus et al., J. Immunol. Methods, 341: 154-64, 2009; and Fujisaki etal., Cancer Res. 69: 4010-4017, 2009). In some embodiments,representative methods of determining B-cell activation can be found inBiological Example 3 provided herein. In some embodiments,representative methods of determining NK-cell activation can be found inBiological Example 3 provided herein.

Modulation of activity of an immune cell, such as a T-cell, a B-cell ora NK-cell can be measured by determining a baseline value for aparameter of interest (e.g., cytokine secretion). For example, T-cellactivation, such as in a sample obtained from in vitro experiments ofcells contacted with a Cbl-b inhibitor, can be measured beforecontacting or administering said Cbl-b inhibitor to determine a baselinevalue. A reference value then is obtained for T-cell activation aftercontacting or administering said Cbl-b inhibitor. The reference value iscompared to the baseline value in order to determine the amount ofT-cell activation due to contact or administration of the Cbl-binhibitor or composition thereof. For example, in some embodiments,immune cell (e.g., T-cell) activation is increased by at least 0.1-foldin a sample as compared to a baseline value, wherein the baseline valueis obtained before contacting the immune cell (e.g., T-cell) with aCbl-b inhibitor or a composition thereof. In some embodiments, immunecell (e.g., T-cell) activation is increased by at least about 0.1-fold,about 0.2-fold, about 0.3-fold, about 0.4-fold, about 0.5-fold, about0.6-fold, about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1-fold,about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold,about 20-fold, about 30-fold, about 40-fold, about 50-fold, about75-fold, or about 100-fold over a baseline value (e.g., about 0.1-foldto about 100-fold, or about 1-fold to about 100-fold). Immune cellactivation can be assessed by measuring biological markers of activationsuch as increased cytokine secretion, increased cell surface expressionof activation markers (e.g., cell surface markers), or increasedphosphorylation of proteins in a downstream signaling pathway. The foldover baseline value that indicates immune cell activation can bedetermined for the parameter being tested and the conditions under whichthe immune cells are treated. For example, for measuring T-cellactivation, a baseline value can be obtained from T-cells stimulatedwith anti-CD3 antibody in combination with anti-CD28 antibody, whereinthe cells are not incubated with a Cbl-b inhibitor. A reference value isthen obtained from T-cells stimulated with anti-CD3 antibody incombination with anti-CD28 antibody, wherein the T-cells have been orare in contact with a Cbl-b inhibitor. A positive response for immunecell activation can then be determined by the obtained reference value.Similar reference value measurements can be obtained and compared to abaseline value for assessing T-cell activation, T-cell proliferation,T-cell exhaustion, T-cell tolerance, B-cell activation and/or NK-cellactivation. Measurements for these parameters can be obtained utilizingtechniques well known in the art, as well as the techniques provided inBiological Examples 2 and 3.

The terms “baseline” or “baseline value” as used herein can refer to ameasurement or characterization before administration of a therapeuticagent as disclosed herein (e.g., a composition comprising a Cbl-binhibitor as described herein) or at the beginning of administration ofthe therapeutic agent. The baseline value can be compared to a referencevalue in order to determine the increase or decrease of an immune cellfunction (e.g., increasing T-cell activation, increasing T-cellproliferation, decreasing T-cell exhaustion, and/or decreasing T-celltolerance). The terms “reference” or “reference value” as used hereincan refer to a measurement or characterization after administration ofthe therapeutic agent as disclosed herein (e.g., a compositioncomprising a Cbl-b inhibitor as described herein). The reference valuecan be measured one or more times during an experimental time course,dosage regimen, or treatment cycle, or at the completion of theexperimental time course, dosage regimen, or treatment cycle. A“reference value” can be an absolute value, a relative value, a valuethat has an upper and/or lower limit, a range of values, an averagevalue, a median value, a mean value, or a value as compared to abaseline value. Similarly, a “baseline value” can be an absolute value,a relative value, a value that has an upper and/or lower limit, a rangeof values, an average value, a median value, a mean value, or a value ascompared to a reference value. The reference value and/or baseline valuecan be obtained from one sample (e.g., one sample obtained from anindividual), from two different samples (e.g., a sample obtained fromtwo different individuals) or from a group of samples (e.g., samplesobtained from a group of two, three, four, five, or more individuals).

In some embodiments, a positive response for T-cell activation asmeasured by cytokine secretion (e.g., IL-2 secretion) by T-cellsstimulated with anti-CD3 antibody in combination with anti-CD28 antibodyin the presence of a Cbl-b inhibitor is at least 2.5-fold over thebaseline value for cytokine secretion (e.g., IL-2 secretion) obtainedfrom T-cells stimulated with anti-CD3 antibody in combination withanti-CD28 antibody in the absence of a Cbl-b inhibitor. In someembodiments, a positive response for T-cell activation as measured bysurface marker expression (e.g., CD25 surface marker staining) byT-cells stimulated with anti-CD3 antibody in combination with anti-CD28antibody in the presence of a Cbl-b inhibitor is at least 1.3-fold overthe baseline value for surface marker expression (e.g., CD25 surfacemarker staining) obtained from T-cells stimulated with anti-CD3 antibodyin combination with anti-CD28 antibody in the absence of a Cbl-binhibitor. In some embodiments, a baseline value can be obtained fromT-cells stimulated with anti-CD3 antibody alone, wherein the cells arenot incubated with a Cbl-b inhibitor. In some embodiments, a positiveresponse for T-cell activation as measured by cytokine secretion (e.g.,IL-2 secretion) by T-cells stimulated with anti-CD3 antibody alone inthe presence of a Cbl-b inhibitor is at least 0.1-fold over the baselinevalue for cytokine secretion (e.g., IL-2 secretion) obtained fromT-cells stimulated with anti-CD3 antibody alone in the absence of aCbl-b inhibitor. In some embodiments, a positive response for T-cellactivation as measured by surface marker expression (e.g., CD25 surfacemarker staining) by T-cells stimulated with anti-CD3 antibody alone inthe presence of a Cbl-b inhibitor is at least 0.6-fold over the baselinevalue for surface marker expression (e.g., CD25 surface marker staining)obtained from T-cells stimulated with anti-CD3 antibody alone in theabsence of a Cbl-b inhibitor.

In some aspects, provided herein are methods of producing a modifiedimmune cell, comprising culturing a cell population containing an immunecell in the presence of an effective amount of a Cbl-b inhibitorprovided herein or a composition thereof to modulate the activity of theimmune cell, thereby producing the modified immune cell. In someembodiments, the immune cell is a T-cell, a B-cell, or a natural killer(NK) cell.

In some embodiments of the methods for producing a modified immune cell,the immune cell that is to be modified is a cell selected from the groupconsisting of: a hematopoietic cell, a multipotent stem cell, a myeloidprogenitor cell, a lymphoid progenitor cell, a T-cell, a B-cell, and aNK-cell. In some embodiments, the method further comprises culturing theimmune cell with stimulating agents such as cytokines or antibodies thatbind to activating proteins expressed by the immune cell (e.g., ananti-CD3 antibody and/or an anti-CD28 antibody). In some embodiments,the immune cell that is to be modified is in a cell populationcontaining the immune cell, wherein the cell population is obtained as asample from an individual. In some embodiments, the immune cell that isto be modified is in a cell population containing the immune cell,wherein the cell population is obtained from culturing a biologicalsample (e.g., blood sample, bone marrow sample, etc.) from anindividual. In some embodiments, the immune cell is modified bycontacting the cell population containing the immune cell with a Cbl-binhibitor or composition thereof thereby producing a modified immunecell. In some embodiments, the modified immune cell is a cell selectedfrom the group consisting of: a hematopoietic cell, a multipotent stemcell, a myeloid progenitor cell, a lymphoid progenitor cell, a T-cell, aB-cell, and a NK-cell. In some embodiments, the immune cell is the samecell type as the modified immune cell. For example, the immune cell canbe an inactive T-cell and the modified immune cell can be an activatedT-cell. In some embodiments, the immune cell is a different cell typethan the modified immune cell. For example, the immune cell can be ahematopoietic stem cell and the modified immune cell can be an NK-cellthat has differentiated from the hematopoietic stem cell. In someembodiments of the method of producing the modified immune cell, themethod further comprises recovering the modified immune cell. In someembodiments, the cell population containing the immune cell, the immunecell or the modified immune cell is from an individual (e.g., a human).In some embodiments, the immune cell or modified immune cell is a humanimmune cell or human modified immune cell, respectively.

Further provided herein are modified immune cells produced by any of themethods described herein such as culturing a cell population containingan immune cell in the presence of an effective amount of a Cbl-binhibitor to modulate the activity of the immune cell and therebyproduce the modified immune cell.

In some embodiments, the Cbl-b inhibitors provided herein are cellmembrane permeable. Accordingly, in some embodiments, a modified immunecell provided herein can comprise a Cbl-b inhibitor described hereinsuch as in the cytoplasm of the modified immune cell.

In some aspects, provided herein is an isolated modified immune cell,wherein the modified immune cell has been contacted or is in contactwith a Cbl-b inhibitor described herein or a composition thereof. Insome embodiments, the modified immune cell is a T-cell, a B-cell, or anatural killer (NK) cell. In some embodiments, the modified immune cellis a hematopoietic cell, a multipotent stem cell, a myeloid progenitorcell, a lymphoid progenitor cell, a T-cell, a B-cell, or a NK-cell.

In some embodiments of the isolated modified immune cell, the modifiedimmune cell is a T-cell, and the T-cell exhibits increased T-cellactivation, increased T-cell proliferation, decreased T-cell exhaustion,and/or decreased T-cell tolerance. In some embodiments, increased T-cellactivation comprises increased production of one or more cytokines fromT-cells or surrounding immune cells in the activated T-cellmicroenvironment (e.g., myeloid cells). In some embodiments, the one ormore cytokines include, but are not limited to: IFN-γ, IL-1β, IL-2,IL-4, IL-5, IL-6, IL-13, IL-18, TNFα, and GM-CSF. In some embodiments,the one or more cytokines is one or more selected from the groupconsisting of: IL-2, IFN-γ, TNFα, and GM-CSF. In some embodiments, thecytokine is a chemokine. In some embodiments, the one or more chemokinesinclude, but are not limited to: IP-10, Eotaxin, GRO alpha, RANTES,MIP-1a, MIP-1β, MIP-2, MCP-1, and MCP-3. In some embodiments, increasedT-cell activation comprises increased cell surface expression of one ormore T-cell activation markers. In some embodiments, the one or moreT-cell activation markers include, but are not limited to: CD25, CD44,CD62L, CD69, CD152 (CTLA4), CD154, CD137, and CD279. In someembodiments, the one or more T-cell activation markers include, but arenot limited to: CD25, CD69, and CTLA4. In some embodiments, the T-cellactivation markers are CD25 and/or CD69. In some embodiments, the T-cellhas been or is in contact with an anti-CD3 antibody. In someembodiments, the T-cell has been or is in contact with an anti-CD3antibody in combination with an anti-CD28 antibody.

In some embodiments of the isolated modified immune cell, the modifiedimmune cell is a NK-cell, and the NK-cell exhibits increased NK-cellactivation. In some embodiments, increased NK-cell activation comprisesincreased secretion of one or more cytokines (e.g., IFN-γ, TNFα, and/orMIP-1β). In some embodiments, increased NK-cell activation comprisesincreased cell surface expression of one or more NK-cell activationmarkers (e.g., CD69 and/or CD107a).

In some embodiments of the isolated modified immune cell, the modifiedimmune cell is a B-cell, and the B-cell exhibits increased B-cellactivation. In some embodiments, increased B-cell activation comprisesincreased cell surface expression of one or more B-cell activationmarkers (e.g., CD69, CD86, and/or HLA-DR).

In some of any embodiments of the methods or modified immune cellsprovided herein, the immune cell or modified immune cell is a mammaliancell (e.g., human cell). In some embodiments, the immune cell ormodified immune cell is a human cell.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177; Klebanoff et al., JImmunother., 35: 651-660, 2012; Terakura et al., Blood, 119: 72-82,2012; and Wang et al., J Immunother., 35:689-701, 2012.

The immune cells to be modified or modified immune cells provided hereincan be engineered to express a recombinant chimeric receptor such as achimeric antigen receptor (CAR). In some embodiments, the CAR comprisesfrom its N terminus to C terminus: an extracellular ligand-bindingdomain, a transmembrane domain, an intracellular costimulatory domain,and an activating cytoplasmic signaling domain. In some embodiments, theCAR comprises from its N terminus to C terminus: an extracellularligand-binding domain, a transmembrane domain, and an activatingcytoplasmic signaling domain. The immune cells can be engineered toexpress the recombinant chimeric receptor (e.g., CAR) before, during, orafter contact with a Cbl-b inhibitor provided herein. In someembodiments, an immune cell to be modified is a T-cell (e.g., a CD4⁺T-cell or a CD8⁺ T-cell). In a further embodiment, the T-cell comprisesa recombinant chimeric receptor such as a CAR. In some embodiments, themodified immune cell is a modified T-cell (e.g., a CD4⁺ T-cell or a CD8⁺T-cell). In a further embodiment, the modified T-cell comprises arecombinant chimeric receptor such as a CAR. Methods for producingimmune cells expressing recombinant chimeric receptors are well known inthe art such as by the introduction of a nucleic acid encoding therecombinant chimeric receptor (e.g., CAR) to an immune cell (e.g.,T-cell) via a vector (e.g., viral vector). See, for example, seeInternational Patent Application No. WO 2017/096329 and U.S. PublicationNo. US 2017/0204372.

In particular, the present disclosure provides methods of producing anexpanded population of lymphocytes, the method comprising: (a) obtaininga biological sample comprising lymphocytes from an individual withcancer, wherein the individual has received or is receiving an effectiveamount of a Cbl-b inhibitor as a monotherapy or as part of a combinationtherapy, and (b) culturing the lymphocytes in cell culture mediumcomprising at least one T-cell growth factor to produce an expandedpopulation of lymphocytes. In some embodiments, the lymphocytes aretumor infiltrating lymphocytes (TILs). In some embodiments, thelymphocytes are TILs that have been or are isolated from a tumor of amammalian subject with cancer. In other embodiments, the lymphocytes areperipheral blood mononuclear cells (PBMCs). In some embodiments, the atleast one T-cell growth factor comprises one or more of the groupconsisting of IL-2, IL-7, IL-15, and IL-21, optionally wherein the atleast one T-cell growth factor comprises IL-2. In some embodiments, thecell culture medium further comprises an anti-CD3 antibody, or both ananti-CD3 antibody and an anti-CD28 antibody. In some embodiments, thecell culture medium further comprises the Cbl-b inhibitor. In someembodiments, the cell culture medium further comprises irradiated feedercells. In some embodiments, the individual is a human patient. Alsoprovided herein are compositions comprising the expanded population ofTILs produced by the aforementioned methods, and a physiologicallyacceptable buffer.

In some embodiments, methods for isolation and processing of immunecells to be modified or which have been modified (i.e., modified immunecells) include steps for freezing (e.g., cryopreserving) the cells,either before or after isolation, incubation (e.g., incubation with aCbl-b inhibitor), and/or engineering (e.g., introduction of a nucleicacid encoding a recombinant chimeric receptor to the immune cell). Avariety of freezing solutions and parameters known in the art may beused.

B. Adoptive Cell Therapy

The modified immune cells, such as an expanded population of lymphocytesor compositions thereof produced by the methods described herein, can beused as a therapeutic agent in methods of treatment of an individual inneed thereof, such as an individual having cancer. Such methods oftreatment include adoptive cell therapy. In some embodiments, the methodof treatment includes isolating cells from an individual, preparing,processing, culturing, and/or engineering them, as described herein, andre-introducing them into the same individual, before or aftercryopreservation. In some embodiments, the method of treatment includesisolating cells from an individual, preparing, processing, culturing,and/or engineering them, as described herein, and re-introducing theminto a different individual, before or after cryopreservation.

Accordingly, in some aspects, provided herein is a method of modulatingthe immune response in an individual, the method comprisingadministering an effective amount of a modified immune cell describedherein or a composition thereof to an individual in need thereof (e.g.,an individual with a T-cell dysfunction disorder). In some embodiments,the individual has a cancer. In some embodiments, provided herein is amethod of treating a cancer responsive to inhibition of Cbl-b activity,the method comprising administering an effective amount of a modifiedimmune cell described herein or a composition thereof to an individualhaving the cancer responsive to inhibition of Cbl-b activity. In someembodiments, provided herein is a method of inhibiting abnormal cellproliferation, the method comprising administering an effective amountof a modified immune cell described herein or a composition thereof toan individual in need thereof. The term “abnormal cell proliferation” asused herein includes hyperplasia or cancer cell proliferation. Thecancer cell may be derived from a hematologic cancer or anon-hematologic cancer. In some embodiments, the cancer is a hematologiccancer, such as lymphoma, a leukemia, or a myeloma. In otherembodiments, the cancer cell is derived from a non-hematologic cancer,such as a sarcoma, a carcinoma, or a melanoma.

In certain embodiments, an individual in need of treatment, such as anindividual having cancer or a T-cell dysfunction disorder, isadministered a composition comprising the modified immune cells providedherein at a range of about one million to about 100 billion cells, suchas, e.g., 1 million to about 50 billion cells (e.g., about 5 millioncells, about 25 million cells, about 500 million cells, about 1 billioncells, about 5 billion cells, about 20 billion cells, about 30 billioncells, about 40 billion cells, or a range defined by any two of theforegoing values), such as about 10 million to about 100 billion cells(e.g., about 20 million cells, about 30 million cells, about 40 millioncells, about 60 million cells, about 70 million cells, about 80 millioncells, about 90 million cells, about 10 billion cells, about 25 billioncells, about 50 billion cells, about 75 billion cells, about 90 billioncells, or a range defined by any two of the foregoing values), and insome cases about 100 million cells to about 50 billion cells (e.g.,about 120 million cells, about 250 million cells, about 350 millioncells, about 450 million cells, about 650 million cells, about 800million cells, about 900 million cells, about 3 billion cells, about 30billion cells, about 45 billion cells) or any value in between theseranges.

The modified immune cells and compositions thereof are administeredusing standard administration techniques, formulations, and/or devices.Provided are formulations and devices, such as syringes and vials, forstorage and administration of the compositions. Formulations orpharmaceutical compositions comprising the modified immune cells includethose for intravenous, intraperitoneal, subcutaneous, or intramuscularadministration. In some embodiments, the modified immune cells areadministered parenterally. The term “parenteral,” as used herein,includes but is not limited to intravenous, intramuscular, subcutaneous,and intraperitoneal administration. In some embodiments, the cellpopulations are administered to a subject using peripheral systemicdelivery by intravenous, intraperitoneal, or subcutaneous injections.Compositions of the modified immune cells can be provided as sterileliquid preparations, e.g., isotonic aqueous solutions, suspensions,emulsions, dispersions, or viscous compositions, which may in someaspects be buffered to a selected pH. Viscous compositions can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol) and suitable mixtures thereof. Sterile injectable solutions canbe prepared by incorporating the modified immune cells in a solvent,such as in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose, dextrose, or the like.

In some embodiments, the modified immune cells are co-administered withone or more additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. For instance, in some therapeutic regimens of the presentdisclosure, both the modified immune cells and a Cbl-b inhibitor areadministered to a mammalian subject in need thereof, wherein the Cbl-binhibitor is a compound of Formula (I), (I-A)-(I-N), (II-A)-(II-L),(III-A)-(III-D), (IV-A)-(IV-C), (V-A)-(V-G), or (VI-A)-(VI-D), or anyvariation thereof. Thus, in some embodiments the therapeutic regimenscomprise both adoptive cell therapy and chemotherapy.

After the modified immune cells are administered to an individual (e.g.,a human), the biological activity of the modified immune cellpopulations can be measured by methods known in the art. Parameters toassess include specific binding of modified immune cell or other immunecell to antigen, in vivo (e.g., by imaging) or ex vivo (e.g., by ELISAor flow cytometry). In some embodiments, the ability of modified immunecells to destroy target cells can be measured using a cytotoxicity assay(see, e.g., Kochenderfer et al., J. Immunotherapy, 32: 689-702, 2009;and Herman et al., J. Immunological Methods, 285: 25-40, 2004). In someembodiments, the biological activity of the modified immune cells alsocan be measured by assaying expression and/or secretion of certaincytokines, such as IL-2 and IFNγ.

C. Administration of Cbl-b Inhibitor

In some aspects, a Cbl-b inhibitor or composition thereof can beadministered directly to an individual to modulate an immune response,treat a disease or condition (e.g., cancer and/or abnormal cellproliferation) and/or inhibit Cbl-b activity in the individual. TheCbl-b inhibitor may be a compound of Table 1 and/or Table 1A, a tautomerthereof, stereoisomer thereof, or a pharmaceutically acceptable saltthereof.

In some embodiments, provided herein is a method of modulating theimmune response, the method comprising administering an effective amountof a Cbl-b inhibitor provided herein or a composition thereof to anindividual to modulate the immune response in the individual. In someembodiments, the individual has a cancer.

In some embodiments, provided herein is a method of treating cancerresponsive to inhibition of Cbl-b activity, the method comprisingadministering an effective amount of a Cbl-b inhibitor provided hereinor a composition thereof to an individual to treat the cancer responsiveto inhibition of Cbl-b activity.

In some embodiments, provided herein is a method of inhibiting abnormalcell proliferation (e.g., hyperplasia), the method comprisingadministering an effective amount of a Cbl-b inhibitor provided hereinor a composition thereof to an individual to inhibit abnormal cellproliferation in the individual.

In some embodiments, provided herein is a method of inhibiting Cbl-bactivity, the method comprising administering an effective amount of aCbl-b inhibitor provided herein or a composition thereof to anindividual to inhibit Cbl-b activity in the individual.

In some embodiments, such as in the modulation of an immune response inan individual in need thereof (e.g., an individual with a T-celldysfunction disorder), treatment of a disease or condition in anindividual (e.g., an individual cancer and/or abnormal cellproliferation) and/or inhibition of Cbl-b activity in an individual, theappropriate dosage of an active agent, will depend on the type ofcondition, disease, or disorder to be treated, as defined above, theseverity and course of the condition, disease, or disorder, whether theagent is administered for preventive or therapeutic purposes, previoustherapy, the subject's clinical history and response to the Cbl-binhibitor, and the discretion of the attending physician. The Cbl-binhibitor or composition thereof is suitably administered to theindividual at one time or over a series of treatments. In someembodiments, the treatment includes multiple administrations of theCbl-b inhibitor or composition thereof, wherein the interval betweenadministrations may vary. For example, the interval between the firstadministration and the second administration is about one month, and theintervals between the subsequent administrations are about three months.In some embodiments, the Cbl-b inhibitor is administered at a flat dose.In some embodiments, the Cbl-b inhibitor is administered to anindividual at a fixed dose based on the individual's weight (e.g.,mg/kg).

In some aspects of the present disclosure, the cancer is a hematologiccancer. For example, the hematologic cancer may be a lymphoma, aleukemia, or a myeloma. In other aspects of the present disclosure, thecancer is a non-hematologic cancer. In particular, the non-hematologiccancer may be a carcinoma, a sarcoma, or a melanoma.

In some embodiments, the Cbl-b inhibitor is co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. For instance, in some therapeutic regimens of the presentdisclosure, both the Cbl-b inhibitor and modified immune cells areadministered to a mammalian subject in need thereof, wherein the Cbl-binhibitor is a compound of formula (I), (I-A)-(I-N), (II-A)-(II-L),(III-A)-(III-D), (IV-A)-(IV-C), (V-A)-(V-G), or (VI-A)-(VI-D), or anyvariation thereof. The Cbl-b inhibitor may be a compound of Table 1and/or Table 1A, a tautomer thereof, stereoisomer thereof, or apharmaceutically acceptable salt thereof. Thus, in some embodiments thetherapeutic regimens comprise both adoptive cell therapy andchemotherapy.

In some embodiments, the effectiveness of Cbl-b inhibitor administrationin the methods herein (e.g., method of modulating an immune response inan individual) can be assessed by measuring the biological activity ofimmune cells present in a sample (e.g., blood sample) isolated from thetreated individual. For example, the ability of immune cells isolatedfrom the individual after treatment with a Cbl-b inhibitor to destroytarget cells in a cytotoxicity assay may be measured to assess treatmentefficacy. In some embodiments, the biological activity of immune cellspresent in a sample (e.g., blood sample) can be measured by assayingexpression and/or secretion of certain cytokines, such as IL-2 and IFNγ.

The present disclosure provides methods of treating cancer, comprising:administering to an individual with cancer an effective amount of aCbl-b inhibitor, and administering to the individual an effective amountof an additional therapeutic agent. Also provided are methods oftreating an individual with cancer, comprising: administering to theindividual an effective amount of a Cbl-b inhibitor; and administeringto the individual an effective amount of an additional therapeuticagent. Additionally, the present disclosure provides methods ofincreasing an anti-cancer immune response, comprising: administering toan individual with cancer an effective amount of a Cbl-b inhibitor, andadministering to the individual an effective amount of an additionaltherapeutic agent. Further provided are methods of treating cancer,comprising: administering to an individual with cancer an effectiveamount of a Cbl-b inhibitor, wherein the individual has received or isreceiving an effective amount of an additional therapeutic agent.

In some embodiments of the methods of the preceding paragraph, the Cbl-binhibitor and the additional therapeutic agent are administeredconsecutively in either order. As used herein, the terms“consecutively,” “serially,” and “sequentially” refer to administrationof a Cbl-b inhibitor after an additional therapeutic agent, oradministration of the additional therapeutic agent after the Cbl-binhibitor. For instance, consecutive administration may involveadministration of the Cbl-b inhibitor in the absence of the additionaltherapeutic agent during an induction phase (primary therapy), which isfollowed by a post-induction treatment phase comprising administrationof the additional therapeutic agent. The methods may further comprise amaintenance phase comprising administration of the Cbl-b inhibitor orthe additional therapeutic agent. Alternatively, consecutiveadministration may involve administration of the additional therapeuticagent in the absence of the Cbl-b inhibitor during an induction phase(primary therapy), which is followed by a post-induction treatment phasecomprising administration of the Cbl-b inhibitor. The methods mayfurther comprise a maintenance phase comprising administration of theCbl-b inhibitor or the additional therapeutic agent.

In some embodiments of the combination therapy methods, the Cbl-binhibitor and the additional therapeutic agent are administeredconcurrently. As used herein, the terms “concurrently,”“simultaneously,” and “in parallel” refer to administration of a Cbl-binhibitor and an additional therapeutic agent during the same doctorvisit or during the same phase of treatment. For instance, both theCbl-b inhibitor and the additional therapeutic agent may be administeredduring one or more of an induction phase, a treatment phase, and amaintenance phase. However, concurrent administration does not requirethat the Cbl-b inhibitor and the additional therapeutic agent be presenttogether in a single formulation or pharmaceutical composition, or thatthe Cbl-b inhibitor and the additional therapeutic agent be administeredat precisely the same time.

1. Combination Therapy Comprising a Cbl-b Inhibitor and an ImmuneCheckpoint Inhibitor

In some embodiments of the combination therapy methods of the presentdisclosure for treating cancer, the additional therapeutic agentcomprises an immune checkpoint inhibitor. The term “immune checkpoint”refers to a signaling pathway that prevents activation of immune cells,while the term “immune checkpoint inhibitor” refers to a compound thatimpedes the immune checkpoint to remove the brake on activation ofimmune cells. In some embodiments, the immune checkpoint inhibitor is anantagonist of at least one inhibitory checkpoint molecule. In someembodiments, the inhibitory checkpoint molecule is selected from thegroup consisting of PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD125), LAG3(CD223), PVR (CD155), PVRL2 (CD112), PVRL3 (CD113), TIGIT, TIM3 (CD366),and VISTA. In some embodiments, the immune checkpoint inhibitor is anantagonist of at least one inhibitory checkpoint molecule selected fromthe group consisting of PD-1 (CD279), PD-L1 (CD274), and CTLA-4 (CD152).

PD-1 refers to programmed cell death protein 1 (PD-1). PD-1 antagonistssuitable for the treatment methods, medicaments, and uses of the presentdisclosure include any chemical compound or biological molecule thatblocks binding of PD-L1 expressed on a cancer cell or antigen presentingcell to PD-1 expressed on a lymphocyte (T-cell, B-cell, and/or NK-cell).Alternative names or synonyms for PD-1 and its ligand include: CD279,PDCD1, PD1, and SLEB2 for PD-1; and CD274, PDCD1L1, PDL1, B7H1, B7-4,and B7-H for programmed cell death 1 ligand 1 (PD-L1). In someembodiments in which a human subject is being treated, the PD-1antagonist blocks binding of human PD-L1 to human PD-1. The amino acidsequence of the mature form of human PD-1 is set forth as residues21-288 in NCBI Locus No. NP_005009. The amino acid sequence of themature form of human PD-L1 is set forth as residues 19-290 in NCBI LocusNo. NP_054862.

CTLA-4 refers to cytotoxic T-lymphocyte associated protein 4. CTLA-4antagonists suitable for the treatment methods, medicaments, and uses ofthe present disclosure include any chemical compound or biologicalmolecule that blocks binding of CTLA-4 expressed on a lymphocyte(T-cell, B-cell, and/or NK-cell) to a ligand (CD80 and/or CD86)expressed on an antigen presenting cell. Alternative names or synonymsfor CTLA-4 include: CD152, CTLA4, ALPS5, CELIAC3, GRD4, GSE, and IDDM12.In some embodiments in which a human subject is being treated, theCTLA-4 antagonist blocks binding of human CTLA-4 to a human ligand. Theamino acid sequence of the mature form of human CTLA-4 is set forth asresidues 36-223 in NCBI Locus No. NP_005205.

LAG3 refers to lymphocyte activating gene 3 protein. LAG3 antagonistssuitable for the treatment methods, medicaments, and uses of the presentdisclosure include any chemical compound or biological molecule thatblocks binding of LAG3 expressed on a lymphocyte (T-cell, B-cell, and/orNK-cell) to a ligand (MHC class II) expressed on an antigen presentingcell. LAG3 is also known as CD223. In some embodiments in which a humansubject is being treated, the LAG3 antagonist blocks binding of humanLAG3 to a human ligand. The amino acid sequence of the mature form ofhuman LAG3 is set forth as residues 23-525 in NCBI Locus No. NP_002277.

PVR refers to poliovirus receptor. PVR antagonists suitable for thetreatment methods, medicaments, and uses of the present disclosureinclude any chemical compound or biological molecule that blocks bindingof PVR expressed on a cancer cell or an antigen presenting cell to TIGITexpressed on a lymphocyte (T-cell, B-cell, and/or NK-cell). Alternativenames or synonyms for PVR include CD155, PVS, HVED, NECL5, nectin-likeprotein 5, and TAGE4. In some embodiments in which a human subject isbeing treated, the PVR antagonist blocks binding of human PVR to humanTIGIT. There are multiple isoforms of human PVR. The amino acid sequenceof alpha isoform of human PVR is set forth in NCBI Locus No. NP_006496.The amino acid sequence of beta isoform of human PVR is set forth inNCBI Locus No. NP_001129240. The amino acid sequence of gamma isoform ofhuman PVR is set forth in NCBI Locus No. NP_001129241. The amino acidsequence of delta isoform of human PVR is set forth in NCBI Locus No.NP_001129242.

PVRL2 refers to poliovirus receptor related 2. PVRL2 antagonistssuitable for the treatment methods, medicaments, and uses of the presentdisclosure include any chemical compound or biological molecule thatblocks binding of PVRL2 expressed on a cancer cell or an antigenpresenting cell to TIGIT expressed on a lymphocyte (T-cell, B-cell,and/or NK-cell). Alternative names or synonyms for PVRL2 include: CD112,NECTIN2, HVEB, herpesvirus entry mediator B, PRR2, and PVRR2. In someembodiments in which a human subject is being treated, the PVRL2antagonist blocks binding of human PVRL2 to human TIGIT. The amino acidsequence of the alpha isoform of human PVRL2 is set forth in NCBI LocusNo. NP_002847. The amino acid sequence of the delta isoform of humanPVRL2 is set forth in NCBI Locus No. NP_001036189.

PVRL3 refers to poliovirus receptor related 3. PVRL3 antagonistssuitable for the treatment methods, medicaments, and uses of the presentdisclosure include any chemical compound or biological molecule thatblocks binding of PVRL3 expressed on a cancer cell or an antigenpresenting cell to TIGIT expressed on a lymphocyte (T-cell, B-cell,and/or NK-cell). Alternative names or synonyms for PVRL3 include: CD113,NECTIN3, PRR3, and PVRR3. In some embodiments in which a human subjectis being treated, the PVRL3 antagonist blocks binding of human PVRL3 tohuman TIGIT. The amino acid sequence of isoform 1 of human PVRL3 is setforth in NCBI Locus No. NP_056295. The amino acid sequence of isoform 2of human PVRL3 is set forth in NCBI Locus No. NP_001230215. The aminoacid sequence of isoform 3 of human PVRL3 is set forth in NCBI Locus No.NP_001230217.

TIGIT refers to T-cell immunoreceptor with Ig and ITIM domains protein.TIGIT antagonists suitable for the treatment methods, medicaments, anduses of the present disclosure include any chemical compound orbiological molecule that blocks binding of TIGIT expressed on alymphocyte (T-cell, B-cell, or NK-cell) to a ligand (CD112, CD113,and/or CD155) expressed on a cancer cell or an antigen presenting cell.Alternative names or synonyms for TIGIT include: VSIG9, V-set andimmunoglobulin domain containing 9, VSTM3, V-set and transmembranedomain containing 3, and Washington University cell adhesion molecule(WUCAM). In some embodiments in which a human subject is being treated,the TIGIT antagonist blocks binding of human TIGIT to a human ligand.The amino acid sequence of the mature form of human TIGIT is set forthas residues 22-244 in NCBI Locus No.: NP_776160.

TIM3 refers to T-cell immunoglobulin and mucin-domain containing-3protein. TIM3 antagonists suitable for the treatment methods,medicaments, and uses of the present disclosure include any chemicalcompound or biological molecule that blocks binding of TIM3 expressed ona lymphocyte (T-cell, B-cell, or NK-cell) to a ligand (galectin-9phosphatidylserine) expressed on an antigen presenting cell. Alternativenames or synonyms for TIM3 include: CD366, HAVCR2, hepatitis A viruscellular receptor 2, KIM3, and SPTCL. In some embodiments in which ahuman subject is being treated, the TIM3 antagonist blocks binding ofhuman TIM3 to a human ligand. The amino acid sequence of the mature formof human TIM3 is set forth as residues 22-301 in NCBI Locus No.NP_116171.

VISTA refers to V-domain Ig suppressor of T-cell activation. VISTAantagonists suitable for the treatment methods, medicaments, and uses ofthe present disclosure include any chemical compound or biologicalmolecule that blocks binding of VISTA expressed on a lymphocyte (T-cell,B-cell, and/or NK-cell) to a ligand expressed on a cancer cell or anantigen presenting cell. Alternative names or synonyms for VISTAinclude: VSIR, V-set immunoregulatory receptor, PD-1H, B7H5, GI24,PP2135, SISP1, and Dies1. In some embodiments in which a human subjectis being treated, the VISTA antagonist blocks binding of human VISTA toa human ligand. The amino acid sequence of the mature form of humanVISTA is set forth as residues 33-311 in NCBI Locus No.: NP_071436.

The immune checkpoint inhibitor may be a biological molecule. Forinstance, the immune checkpoint inhibitor may comprise an antibody orantigen-binding fragment thereof. The antibody or fragment may be amonoclonal antibody (mAb), for example, a human antibody, a humanizedantibody, or a chimeric antibody, and may include a human constantregion. In some embodiments the human constant region is selected fromthe group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, andin certain embodiments, the human constant region is an IgG1 or IgG4constant region. In some embodiments, the antibody or fragment is abispecific antibody. In some embodiments, the antigen-binding fragmentcomprises one of the group consisting of Fab, Fab′-SH, F(ab′)2, scFv,and Fv fragments.

In some embodiments, the at least one inhibitory checkpoint moleculecomprises PD-1. In some embodiments, the immune checkpoint inhibitor isselected from the group consisting of pembrolizumab, nivolumab,cemiplimab, and biosimilars thereof. In one embodiment, the anti-PD-1antibody is pembrolizumab (MK-3475 marketed as KEYTRUDA® by Merck &Co.). In one embodiment, the anti-PD-1 antibody is nivolumab (BMS-936558or MDX-1106, marketed as OPDIVO® by Bristol-Myers Squibb). In oneembodiment, the anti-PD-1 antibody is cemiplimab (REGN2810, Regeneron).In some embodiments, the immune checkpoint inhibitor is a variant ofpembrolizumab, nivolumab, or cemiplimab.

In some embodiments, the at least one inhibitory checkpoint moleculecomprises PD-L1. In some embodiments, the immune checkpoint inhibitor isselected from the group consisting of atezolizumab, avelumab,durvalumab, and biosimilars thereof. In one embodiment, the anti-PD-L1antibody is atezolizumab (marketed as TECENTRIQ® by Genentech, Inc.). Inone embodiment, the anti-PD-L1 antibody is avelumab (marketed asBAVENCIO® by EMD Serono, Inc. and Pfizer, Inc.). In one embodiment, theanti-PD-L1 antibody is durvalumab (MEDI4736 marketed as IMFINZI® byAstraZeneca). In some embodiments, the immune checkpoint inhibitor is avariant of atezolizumab, avelumab, or durvalumab.

In some embodiments, the at least one inhibitory checkpoint moleculecomprises CTLA-4. In some embodiments, the immune checkpoint inhibitoris selected from the group consisting of ipilimumab, tremelimumab, andbiosimilars thereof. In one embodiment, the anti-CTLA4 antibody isipilimumab (MDX-010 or BMS-734016, marketed as YERVOY® by Bristol-MyersSquibb). In one embodiment, the anti-CTLA4 antibody is tremelimumab(ticilimumab, CP-675,206, developed by AstraZeneca). In someembodiments, the immune checkpoint inhibitor is a variant of ipilimumab,or tremelimumab.

In some embodiments, the monoclonal antibody is a “variant” antibodywhich comprises heavy chain and light chain sequences that are identicalto those in the “reference” antibody, except for having three, two, orone conservative amino acid substitutions at positions that are locatedoutside of the light chain CDRs and/or six, five, four, three, two, orone conservative amino acid substitutions that are located outside ofthe heavy chain CDRs (e.g., the variant positions are located in theframework regions or the constant region). In other words, the referenceantibody and the variant antibody comprise identical CDR sequences, butdiffer from each other due to having a conservative amino acidsubstitution at no more than three or six other positions in their fulllength light and heavy chain sequences, respectively. A variant antibodyis substantially the same as a reference antibody with respect to thefollowing properties: binding affinity to the inhibitory checkpointmolecule and ability to block the binding of the inhibitory checkpointmolecule to its ligand.

In other embodiments, the immune checkpoint inhibitor may comprise animmunoadhesin comprising the inhibitory checkpoint molecule bindingdomain of one of its ligands fused to a constant region such as an Fcregion of an immunoglobulin molecule.

As used herein the term “biosimilar” refers to a biological product thatis similar to but without clinically meaningful differences in safetyand effectiveness from a Federal Drug Administration (FDA)-approvedreference product. For instance, there may be differences between abiosimilar product and a reference product in clinically inactivecomponents (e.g., differences in excipients of the formulations, minordifferences in glycosylation, etc.). Clinically meaningfulcharacteristics can be assessed through pharmacokinetic andpharmacodynamic studies. In some embodiments, the biosimilar product isan interchangeable product as determined by the FDA.

2. Combination Therapy Comprising a Cbl-b Inhibitor and anAntineoplastic Agent

In some embodiments of the combination therapy methods herein fortreating cancer, the additional therapeutic agent comprises anantineoplastic agent. As used herein, the terms “anti-neoplastic agent”and “antineoplastic agent” refer to a therapeutic agent classifiedaccording to the Anatomical Therapeutic Chemical Classification System(ATC) code L01 developed by the World Health Organization. In someembodiments, the antineoplastic agent is classified as one of the groupconsisting of a cytotoxic antibiotic (ATC code L01D), a plant alkaloid(ATC code L01C), an antimetabolite (ATC code L01B), an alkylating agent(ATC code L01A), and other antineoplastic agent (ATC code L01X). In someembodiments, the antineoplastic agent is a small molecule drug (e.g.,cancer chemotherapeutic agent) as opposed to a biological molecule.

A cytotoxic antibiotic is a suitable antineoplastic agent for thetreatment methods, medicaments and uses of the present disclosure. Insome embodiments, the cytotoxic antibiotic is selected from the groupconsisting of ixabepilone, mitomycin, plicamycin, bleomycin, pixantrone,amrubicin, valrubicin, pirarubicin, mitoxantrone, idarubicin, zorubicin,aclarubicin, epirubicin, daunorubicin, doxorubicin, and dactinomycin.

A plant alkaloid is a suitable antineoplastic agent for the treatmentmethods, medicaments, and uses of the present disclosure. In someembodiments, the plant alkaloid is selected from the group consisting oftrabectedin, cabazitaxel, paclitaxel poliglumex, docetaxel, paclitaxel,demecolcine, teniposide, etoposide, vintafolide, vinflunine,vinorelbine, vindesine, vincristine, and vinblastine.

An antimetabolite is a suitable antineoplastic agent for the treatmentmethods, medicaments, and uses of the present disclosure. In someembodiments, the antimetabolite is a pyrimidine analog, a purine analog,or a folic acid analog. In some embodiments, the antimetabolite isselected from the group consisting of floxuridine, trifluridine,tegafur, fluorouracil, decitabine, azacitidine, capecitabine,gemcitabine, carmofur, tegafur, fluorouracil, cytarabine, nelarabine,clofarabine, fludarabine, cladribine, tioguanine, mercaptopurine,pralatrexate, pemetrexed, raltitrexed, and methotrexate.

An alkylating agent is a suitable antineoplastic agent for the treatmentmethods, medicaments, and uses of the present disclosure. In someembodiments, the alkylating agent is selected from the group consistingof dacarbazine, temozolomide, pipobroman, mitobronitol, etoglucid,uracil mustard, ranimustine, nimustine, fotemustine, streptozocin,semustine, lomustine, carmustine, carboquone, triaziquone, thiotepa,mannosulfan, treosulfan, busulfan, bendamustine, prednimustine,trofosfamide, ifosfamide, mechlorethamine, melphalan, chlorambucil, andcyclophosphamide.

In other embodiments, the antineoplastic agent comprises an otherantineoplastic agent selected from the group consisting of a platinumcompound (ATC Code L01XA), a methylhydrazine (ATC Code L01XB), asensitizer (ATC Code L01XD), a protein kinase inhibitor (ATC CodeL01XE), and an other agent (ATC Code L01XA).

A platinum compound is a suitable antineoplastic agent for the treatmentmethods, medicaments, and uses of the present disclosure. In someembodiments, the platinum compound is selected from the group consistingof cisplatin, carboplatin, oxaliplatin, satraplatin, and polyplatillen.

3. Combination Therapy Comprising a Cbl-b Inhibitor and RadiationTherapy

The present disclosure provides methods of treating cancer comprising:administering to an individual with cancer an effective amount of aCbl-b inhibitor, and administering to the individual an effective amountof radiation therapy. Also provided are methods of treating anindividual with cancer, comprising: administering to the individual aneffective amount of a Cbl-b, and administering to the individual aneffective amount of radiation therapy. Additionally, the presentdisclosure provides methods of increasing an anti-cancer immuneresponse, comprising: administering to an individual with cancer aneffective amount of a Cbl-b inhibitor, and administering to theindividual an effective amount of radiation therapy. Further providedare methods of treating cancer, comprising: administering to anindividual with cancer an effective amount of a Cbl-b inhibitor, whereinthe individual has received or is receiving an effective amount ofradiation therapy.

In some embodiments, the radiation therapy is external beam radiationtherapy. In other embodiments, the radiation therapy is internalradiation therapy. In some embodiments, the radiation therapy isablative radiation therapy.

In some embodiments, the combination therapy regimen of the presentdisclosure comprises administration of a Cbl-b inhibitor, radiationtherapy, and one or both of an immune checkpoint inhibitor and anantineoplastic agent.

Provided herein are methods for treating cancer, comprisingadministering to an individual with cancer a combination therapycomprising an effective amount of a Cbl-b inhibitor, and an effectiveamount of a cancer vaccine. Also provided are medicaments comprising aCbl-b inhibitor for use in combination with a cancer vaccine fortreating cancer, and medicaments comprising both a Cbl-b inhibitor and acancer vaccine for use in treating cancer. Further provided are uses ofa Cbl-b inhibitor in the manufacture of a medicament for treating cancerin an individual when administered in combination with a cancer vaccine.Further provided are uses of a Cbl-b inhibitor and a cancer vaccine inthe manufacture of a medicament(s) for treating cancer. In someembodiments, the Cbl-b inhibitor is a “small molecule.”

Also provided herein are methods for treating cancer, comprisingadministering to an individual with cancer a combination therapycomprising an effective amount of a Cbl-b inhibitor, and an effectiveamount of an oncolytic virus. Also provided are medicaments comprising aCbl-b inhibitor for use in combination with an oncolytic virus fortreating cancer, and medicaments comprising both a Cbl-b inhibitor andan oncolytic virus for use in treating cancer. Further provided are usesof a Cbl-b inhibitor in the manufacture of a medicament for treatingcancer in an individual when administered in combination with anoncolytic virus. Further provided are uses of a Cbl-b inhibitor and anoncolytic virus in the manufacture of a medicament(s) for treatingcancer. In some embodiments, the Cbl-b inhibitor is a “small molecule.”

In some embodiments of the treatment methods, medicaments and uses ofthe present disclosure, the cancer is a hematologic cancer such aslymphoma, a leukemia or a myeloma. In other embodiments of the treatmentmethods, medicaments and uses of the present disclosure, the cancer is anon-hematologic cancer such as a sarcoma, a carcinoma, or a melanoma.

Hematologic cancers include, but are not limited to, one or moreleukemias such as B-cell acute lymphoid leukemia (“BALL”), T-cell acutelymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL); one or morechronic leukemias including, but not limited to, chronic myelogenousleukemia (CML) and chronic lymphocytic leukemia (CLL); additionalhematologic cancers or hematologic conditions including, but not limitedto, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cellneoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicularlymphoma, hairy cell leukemia, small cell- or a large cell-follicularlymphoma, malignant lymphoproliferative conditions, MALT lymphoma,mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma,myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, and “preleukemia,” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells.

Non-hematologic cancers include but are not limited to, a neuroblastoma,renal cell carcinoma, colon cancer, colorectal cancer, breast cancer,epithelial squamous cell cancer, melanoma, stomach cancer, brain cancer,lung cancer (e.g., NSCLC), pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, prostate cancer, testicularcancer, thyroid cancer, uterine cancer, adrenal cancer, and head andneck cancer.

In some aspects, the effectiveness of administration an activationthreshold reducer or a costimulation requirement reducer, such as aCbl-b inhibitor, in the treatment of a cancer is measured by assessingclinical outcome, such as reduction in tumor size or number of tumors,and/or survival. In some embodiments, “treating cancer” comprisesassessing a patient's response to the treatment regimen according to theResponse Evaluation Criteria in Solid Tumors (RECIST version 1.1) asdescribed (see, e.g., Eisenhauer et al., Eur J Cancer, 45:228-247, 2009;and Nishino et al., Am J Roentgenol, 195: 281-289, 2010). Responsecriteria to determine objective anti-tumor responses per RECIST 1.1include: complete response (CR); partial response (PR); progressivedisease (PD); and stable disease (SD).

Also provided herein are methods for treating cancer, comprisingadministering to an individual with cancer a combination therapycomprising an effective amount of an agent that lowers activationthreshold (activation threshold reducer) of an immune cell (e.g.,T-cell, B-cell, and/or NK-cell), and an effective amount of a cancervaccine. Also provided are medicaments comprising an activationthreshold reducer for use in combination with a cancer vaccine fortreating cancer, and medicaments comprising both an activation thresholdreducer and a cancer vaccine for use in treating cancer. Furtherprovided are uses of an activation threshold reducer in the manufactureof a medicament for treating cancer in an individual when administeredin combination with a cancer vaccine. Further provided are uses of anactivation threshold reducer and a cancer vaccine in the manufacture ofa medicament(s) for treating cancer.

Also provided herein are methods for treating cancer, comprisingadministering to an individual with cancer a combination therapycomprising an effective amount of an agent that lowers activationthreshold (activation threshold reducer) of an immune cell (e.g.,T-cell, B-cell, and/or NK-cell), and an effective amount of an oncolyticvirus. Also provided are medicaments comprising an activation thresholdreducer for use in combination with an oncolytic virus for treatingcancer, and medicaments comprising both an activation threshold reducerand an oncolytic virus for use in treating cancer. Further provided areuses of an activation threshold reducer in the manufacture of amedicament for treating cancer in an individual when administered incombination with an oncolytic virus. Further provided are uses of anactivation threshold reducer and an oncolytic virus in the manufactureof a medicament(s) for treating cancer.

In some embodiments, the agent that lowers activation threshold(activation threshold reducer) is an agent that reduces costimulationrequirement (costimulation requirement reducer) of an immune cell (e.g.,T-cell, B-cell, and/or NK-cell). In some embodiments, the agent thatlowers activation threshold (activation threshold reducer) is an agentthat promotes tumor immune-surveillance. In some embodiments, the agentthat lowers activation threshold (activation threshold reducer) is aCbl-b inhibitor. In some embodiments, the agent that reducescostimulation requirement is a Cbl-b inhibitor. In some embodiments, theagent that promotes tumor immune-surveillance is a Cbl-b inhibitor.

In some embodiments, an activation threshold reducer, such as a Cbl-binhibitor, is capable of increasing T-cell activation and/or T-cellproliferation. In some embodiments, an activation threshold reducer,such as a Cbl-b inhibitor, is capable of decreasing T-cell exhaustion,T-cell tolerance, and/or T-cell anergy.

In some embodiments, an activation threshold reducer, such as a Cbl-binhibitor, is capable of increasing production of one or more cytokinesby T-cells or surrounding immune cells in the activated T-cellmicroenvironment (e.g., myeloid cells). In some embodiments, the one ormore cytokines include, but are not limited to: IFN-γ, IL-1, IL-2, IL-4,IL-5, IL-6, IL-13, IL-18, TNFα, and GM-CSF. In some embodiments, thecytokine is one or more of: IL-2, IFN-γ, TNFα, and GM-CSF. In someembodiments, the cytokine is a chemokine. In some embodiments, the oneor more chemokines include, but are not limited to: IP-10, Eotaxin, GROalpha, RANTES, MIP-1α, MIP-1β, MIP-2, MCP-1, and MCP-3. Increasedexpression of cytokines can be measured by ELISA.

In some embodiments, an activation threshold reducer, such as a Cbl-binhibitor, is capable of increasing cell surface expression of one ormore T-cell activation markers. In some embodiments, the one or moreT-cell activation markers include, but are not limited to: CD25, CD44,CD62L, CD69, CD152 (CTLA4), CD154, CD137, and CD279. In someembodiments, the T-cell activation marker is one or more of: CD25, CD69,and CTLA4. Increased expression of cell surface markers can be measuredby FACS.

Methods for experimentally determining increased T-cell activation,increased T-cell proliferation, decreased T-cell exhaustion, and/ordecreased T-cell tolerance are well known in the art. In someembodiments, representative methods of determining T-cell activation canbe found in Biological Example 9 and/or Biological Example 13 providedherein. In some embodiments, representative in vitro and in vivo methodsof determining increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and/or decreased T-celltolerance can be found in Biological Example 10 and/or BiologicalExample 14.

In some embodiments, an activation threshold reducer, such as a Cbl-binhibitor, is capable of increasing B-cell activation. In someembodiments, increased B-cell activation comprises increased cellsurface expression of one or more B-cell activation markers. In someembodiments, the one or more B-cell activation markers include, but arenot limited to: CD69, CD86, and MHC class II (e.g., HLA-DR). In someembodiments, the B-cell activation marker is CD69. Increased expressionof cell surface markers can be measured by FACS. In some embodiments,increased B-cell activation comprises increased activation of proteinsin signaling pathways, such as those mediated by ERK, INK, and Syk.Increased activation of proteins can be detected by measurement oflevels of phosphorylation on the proteins using reagents, such asanti-phospho antibodies available in the art.

In some embodiments, an activation threshold reducer, such as a Cbl-binhibitor, is capable of increasing NK-cell activation. In someembodiments, increased NK-cell activation comprises secretion of one ormore cytokines. In some embodiments, the one or more cytokines include,but are not limited to: IFN-γ, TNFα, and MIP-1β. Increased expression ofcytokines can be measured by ELISA. In some embodiments, increasedNK-cell activation comprises increased cell surface expression of one ormore NK-cell activation markers. In some embodiments, the one or moreNK-cell activation markers include, but are not limited to: CD69, andCD107a. Increased expression of cell surface markers can be measured byFACS. In some embodiments, increased NK-cell activation comprisesincreased killing of target cells such as tumor cells, including primarytumor cells, and cell line derived tumor cells, such as the K562 cellline.

Methods for experimentally determining increased B-cell activation andNK-cell activation are well known in the art. In some embodiments,representative methods of determining B-cell activation can be found inBiological Example 10 and/or Biological Example 14. In some embodiments,representative methods of determining NK-cell activation can be found inBiological Example 10 and/or Biological Example 14.

Modulation of activity of an immune cell, such as a T-cell, a B-cell, ora NK-cell can be measured by determining a baseline value for aparameter of interest (e.g., cytokine secretion). For example, T-cellactivation, such as in a sample obtained from in vitro experiments ofcells contacted with a Cbl-b inhibitor, can be measured beforecontacting or administering said Cbl-b inhibitor to determine a baselinevalue. A reference value then is obtained for T-cell activation aftercontacting or administering said Cbl-b inhibitor. The reference value iscompared to the baseline value in order to determine the amount ofT-cell activation due to contact or administration of the Cbl-binhibitor or composition thereof. For example, in some embodiments,immune cell (e.g., T-cell) activation is increased by at least 0.1-foldin a sample as compared to a baseline value, wherein the baseline valueis obtained before contacting the immune cell (e.g., T-cell) with aCbl-b inhibitor or a composition thereof. In some embodiments, immunecell (e.g., T-cell) activation is increased by at least about 0.1-fold,about 0.2-fold, about 0.3-fold, about 0.4-fold, about 0.5-fold, about0.6-fold, about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1-fold,about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold,about 20-fold, about 30-fold, but no more than about 50-fold over abaseline value. Immune cell activation can be assessed by measuringbiological markers of activation such as increased cytokine secretion,increased cell surface expression of activation markers (e.g., cellsurface markers), or increased phosphorylation of proteins in adownstream signaling pathway. The fold over baseline value thatindicates immune cell activation can be determined for the parameterbeing tested and the conditions under which the immune cell weretreated. For example, for measuring T-cell activation, a baseline valuecan be obtained from T-cells stimulated with anti-CD3 antibody incombination with anti-CD28 antibody, wherein the cells are not incubatedwith a Cbl-b inhibitor. A reference value is then obtained from T-cellsstimulated with anti-CD3 antibody in combination with anti-CD28antibody, wherein the T-cells have been or are in contact with a Cbl-binhibitor. A positive response for immune cell activation can then bedetermined by the obtained reference value. Similar reference valuemeasurements can be obtained and compared to a baseline value forassessing T-cell activation, T-cell proliferation, T-cell exhaustion,T-cell tolerance, B-cell activation and/or NK-cell activation.Measurements for these parameters can be obtained utilizing techniqueswell known in the art, as well as the techniques provided in BiologicalExamples 9, 10, 12, and 13.

The terms “baseline” or “baseline value” as used herein can refer to ameasurement or characterization before administration of a therapeuticagent as disclosed herein (e.g., a composition comprising a Cbl-binhibitor as described herein) or at the beginning of administration ofthe therapeutic agent. The baseline value can be compared to a referencevalue in order to determine the increase or decrease of an immune cellfunction (e.g., increasing T-cell activation, increasing T-cellproliferation, decreasing T-cell exhaustion, and/or decreasing T-celltolerance). The terms “reference” or “reference value” as used hereincan refer to a measurement or characterization after administration ofthe therapeutic agent as disclosed herein (e.g., a compositioncomprising a Cbl-b inhibitor as described herein). The reference valuecan be measured one or more times during an experimental time course,dosage regimen, or treatment cycle, or at the completion of theexperimental time course, dosage regimen, or treatment cycle. A“reference value” can be an absolute value, a relative value, a valuethat has an upper and/or lower limit, a range of values, an averagevalue, a median value, a mean value, or a value as compared to abaseline value. Similarly, a “baseline value” can be an absolute value,a relative value, a value that has an upper and/or lower limit, a rangeof values, an average value, a median value, a mean value, or a value ascompared to a reference value. The reference value and/or baseline valuecan be obtained from one sample (e.g., one sample obtained from anindividual), from two different samples (e.g., a sample obtained fromtwo different individuals) or from a group of samples (e.g., samplesobtained from a group of two, three, four, five or more individuals).

In some embodiments, a positive response for T-cell activation asmeasured by cytokine secretion (e.g., IL-2 secretion) by T-cellsstimulated with anti-CD3 antibody in combination with anti-CD28 antibodyin the presence of a Cbl-b inhibitor is at least 2.5-fold over thebaseline value for cytokine secretion (e.g., IL-2 secretion) obtainedfrom T-cells stimulated with anti-CD3 antibody in combination withanti-CD28 antibody in the absence of a Cbl-b inhibitor. In someembodiments, a positive response for T-cell activation as measured bysurface marker expression (e.g., CD25 surface marker staining) byT-cells stimulated with anti-CD3 antibody in combination with anti-CD28antibody in the presence of a Cbl-b inhibitor is at least 1.3-fold overthe baseline value for surface marker expression (e.g., CD25 surfacemarker staining) obtained from T-cells stimulated with anti-CD3 antibodyin combination with anti-CD28 antibody in the absence of a Cbl-binhibitor. In some embodiments, a baseline value can be obtained fromT-cells stimulated with anti-CD3 antibody alone, wherein the cells arenot incubated with a Cbl-b inhibitor. In some embodiments, a positiveresponse for T-cell activation as measured by cytokine secretion (e.g.,IL-2 secretion) by T-cells stimulated with anti-CD3 antibody alone inthe presence of a Cbl-b inhibitor is at least 0.1-fold over the baselinevalue for cytokine secretion (e.g., IL-2 secretion) obtained fromT-cells stimulated with anti-CD3 antibody alone in the absence of aCbl-b inhibitor. In some embodiments, a positive response for T-cellactivation as measured by surface marker expression (e.g., CD25 surfacemarker staining) by T-cells stimulated with anti-CD3 antibody alone inthe presence of a Cbl-b inhibitor is at least 0.6-fold over the baselinevalue for surface marker expression (e.g., CD25 surface marker staining)obtained from T-cells stimulated with anti-CD3 antibody alone in theabsence of a Cbl-b inhibitor.

The present disclosure provides methods of treating cancer, comprising:administering to an individual with cancer an effective amount of aCbl-b inhibitor, and administering to the individual an effective amountof a cancer vaccine. Also provided are methods of treating an individualwith cancer, comprising: administering to the individual an effectiveamount of a Cbl-b inhibitor; and administering to the individual aneffective amount of a cancer vaccine. Additionally, the presentdisclosure provides methods of increasing an anti-cancer immuneresponse, comprising: administering to an individual with cancer aneffective amount of a Cbl-b inhibitor, and administering to theindividual an effective amount of a cancer vaccine. Further provided aremethods of treating cancer, comprising: administering to an individualwith cancer an effective amount of a Cbl-b inhibitor, wherein theindividual has received or is receiving an effective amount of a cancervaccine.

In addition, the present disclosure provides methods of treating cancer,comprising: administering to an individual with cancer an effectiveamount of a Cbl-b inhibitor, and administering to the individual aneffective amount of an oncolytic virus. Also provided are methods oftreating an individual with cancer, comprising: administering to theindividual an effective amount of a Cbl-b inhibitor; and administeringto the individual an effective amount of an oncolytic virus.Additionally, the present disclosure provides methods of increasing ananti-cancer immune response, comprising: administering to an individualwith cancer an effective amount of a Cbl-b inhibitor, and administeringto the individual an effective amount of an oncolytic virus. Furtherprovided are methods of treating cancer, comprising: administering to anindividual with cancer an effective amount of a Cbl-b inhibitor, whereinthe individual has received or is receiving an effective amount of anoncolytic virus.

In some embodiments of the methods of the preceding paragraph, the Cbl-binhibitor and the cancer vaccine are administered consecutively ineither order. In certain embodiments, as used herein, the terms“consecutively”, “serially”, and “sequentially” refer to administrationof a Cbl-b inhibitor after a cancer vaccine, or administration of thecancer vaccine after the Cbl-b inhibitor. For instance, consecutiveadministration may involve administration of the Cbl-b inhibitor in theabsence of the cancer vaccine during an induction phase (primarytherapy), which is followed by a post-induction treatment phasecomprising administration of both the cancer vaccine and the Cbl-binhibitor. The methods may further comprise a maintenance phasecomprising administration of the Cbl-b inhibitor or administration of afurther dose of the cancer vaccine. Alternatively, consecutiveadministration may involve administration of the cancer vaccine in theabsence of the Cbl-b inhibitor during an induction phase (primarytherapy), which is followed by a post-induction treatment phasecomprising administration of the Cbl-b inhibitor. The methods mayfurther comprise a maintenance phase comprising administration of theCbl-b inhibitor or a further dose of the cancer vaccine.

In some embodiments of the methods of the preceding paragraph, the Cbl-binhibitor and the oncolytic virus are administered consecutively ineither order. In certain embodiments, as used herein, the terms“consecutively”, “serially”, and “sequentially” refer to administrationof a Cbl-b inhibitor after an oncolytic virus, or administration of theoncolytic virus after the Cbl-b inhibitor. For instance, consecutiveadministration may involve administration of the Cbl-b inhibitor in theabsence of the oncolytic virus during an induction phase (primarytherapy), which is followed by a post-induction treatment phasecomprising administration of both the oncolytic virus and the Cbl-binhibitor. The methods may further comprise a maintenance phasecomprising administration of the Cbl-b inhibitor or administration of afurther dose of the oncolytic virus. Alternatively, consecutiveadministration may involve administration of the oncolytic virus in theabsence of the Cbl-b inhibitor during an induction phase (primarytherapy), which is followed by a post-induction treatment phasecomprising administration of the Cbl-b inhibitor. The methods mayfurther comprise a maintenance phase comprising administration of theCbl-b inhibitor or a further dose of the oncolytic virus.

In some embodiments of the combination therapy methods, the Cbl-binhibitor and the cancer vaccine are administered concurrently. Incertain embodiments, as used herein, the terms “concurrently”,“simultaneously”, and “in parallel” refer to administration of a Cbl-binhibitor and a cancer vaccine during the same doctor visit or duringthe same phase of treatment. For instance, both the Cbl-b inhibitor andthe cancer vaccine may be administered during one or more of aninduction phase, a treatment phase, and a maintenance phase. However,concurrent administration does not require that the Cbl-b inhibitor andthe cancer vaccine be present together in a single formulation orpharmaceutical composition, or that the Cbl-b inhibitor and the cancervaccine be administered at precisely the same time.

In some embodiments of the combination therapy methods, the Cbl-binhibitor and the oncolytic virus are administered concurrently. Incertain embodiment, as used herein, the terms “concurrently”,“simultaneously”, and “in parallel” refer to administration of a Cbl-binhibitor and an oncolytic virus during the same doctor visit or duringthe same phase of treatment. For instance, both the Cbl-b inhibitor andthe oncolytic virus may be administered during one or more of aninduction phase, a treatment phase, and a maintenance phase. However,concurrent administration does not require that the Cbl-b inhibitor andthe oncolytic virus be present together in a single formulation orpharmaceutical composition, or that the Cbl-b inhibitor and theoncolytic virus be administered at precisely the same time.

In some aspects, the treatment includes multiple administrations of theCbl-b inhibitor or composition thereof, wherein the interval betweenadministrations may vary. In some embodiments, the Cbl-b inhibitor isadministered at a flat dose to an individual (e.g., mg/adult ormg/child). In some embodiments, the Cbl-b inhibitor is administered toan individual at a fixed dose based in the individual's weight (e.g.,mg/kg).

In some embodiments, the effectiveness of the combination therapiesdisclosed herein can be assessed by measuring the biological activity ofimmune cells present in a sample isolated from the treated individual.For example, the ability of immune cells, which are isolated from theindividual after treatment, to destroy target cells using a cytotoxicityassay can be used to assess treatment efficacy. In some embodiments, thebiological activity of immune cells present in a sample can be measuredby assaying expression and/or secretion of certain cytokines, such asIL-2 and IFNγ.

The term “cancer vaccine” as used herein, unless otherwise specified,refers to a “therapeutic cancer vaccine” to be administered to anindividual with cancer for the purpose of treating cancer (andoptionally preventing recurrence of the cancer). In contrast, a“preventative cancer vaccine” (prophylactic cancer vaccine) is to beadministered to an individual without cancer for the purpose ofpreventing cancer or reducing the individual's risk of developingcancer. Examples of preventative cancer vaccines are humanpapillomavirus vaccines for prevention squamous cell carcinoma andhepatitis B virus vaccines for prevention of hepatocellular carcinoma.Cancer vaccines are immunogenic compositions comprising apharmaceutically acceptable excipient and at least one tumor antigen,such as a tumor-specific antigen or a tumor-associated antigen.

As used herein, the terms “oncolytic virus” and “OV” refer to a virusthat infects and kills cancer cells. Death of cancer cells is a resultof both direct cytolysis and induction of anti-tumor immunity. In someembodiments, the “oncolytic virus” is a replication-competent virus,which selectively replicates in cancer cells. In other embodiments, the“oncolytic virus” is a replication-deficient virus, which does notreplicate in cancer cells either as a consequence of genetic engineeringor inactivation (e.g., UV-irradiation or heat) of the oncolytic virus.

In some embodiments, the tumor antigen comprises a “shared tumorantigen” that is common to many cancers of the same type. Non-limitingexamples of shared tumor antigens are the breast cancer antigen HER2,the prostate cancer antigens PAP and PSA, and the melanoma antigensMART-1 and MAGE. In other embodiments, the tumor antigen comprises a“neoantigen” that arises as a result of a tumor-specific DNA alteration(e.g., somatic mutation). As such, neoantigens typically possess anamino acid sequence not present in a normal mammalian genome (Schumacherand Schreiber, Science, 348: 69-74, 2015). Non-limiting examples ofneoantigens are BRAF V600E, KRAS G12D, KRAS G12V, PIK3CA H1047R, andPIC3CA E545K. A tumor-specific neoantigen database (TSNAdb) is nowfreely available (Wu et al., Genomics Proteomics Bioinformatics 16:276-282, 2018).

A variety of techniques are suitable for identification of neoantigensfor inclusion in a cancer vaccine as part of a combination therapycomprising an activation threshold reducer, such as a Cbl-b inhibitor.For instance, neoantigens can be identified with methods comprisingisolating DNA from a tumor biopsy obtained from an individual,sequencing the DNA, and computational analysis of the sequence toidentify one or more neoantigens (Aldous and Dong, Bioorg Med Chem, 26:2842-2849, 2018). In some embodiments, the computational analysisinvolves identifying peptides of 8-11 amino acids in length that arepredicted to bind to at least one HLA allele expressed by cells of thetumor and which comprise at least one missense mutation (Wu et al.,Genomics Proteomics Bioinformatics 16: 276-282, 2018). Neoantigeninclusion in a cancer vaccine is thought to be advantageous forovercoming tolerance and reducing autoimmunity risk.

Cancer vaccine platforms suitable for use in the methods, medicaments,and uses of the present disclosure include, but are not limited to,synthetic peptides, recombinant proteins, nucleic acids (DNA or mRNA),microbial vectors, tumor cells, and antigen presenting cells (see, e.g.,DeMaria and Bilusic, Hematol Oncol Cin North Am, 33: 199-214, 2019; andMaeng and Berzofsky, F1000Research 2019, 8(F1000 Faculty Rev): 654,2019).

In some embodiments, the tumor antigen of the cancer vaccine comprisesat least one synthetic peptide or recombinant protein. In someembodiments, the synthetic peptide is at least 8 amino acids in length,and in certain embodiments, less than 80 amino acids in length. In someembodiments, the tumor antigen comprises a plurality of syntheticpeptides, or the tumor antigen comprises a synthetic peptide or arecombinant protein comprising the amino acid sequence of two, three, ormore epitopes. An “epitope” is a portion of an antigen that is bound byan antibody or a B-cell receptor, or that is presented for binding by aT-cell receptor by a major histocompatibility complex molecule (MHCclass I or class II) on the surface of a cell, such as a tumor cell or adendritic cell. In some embodiments, the epitope is a “linear epitope”composed of contiguous amino acids of a tumor antigen sequence (primarystructure). In some embodiments, the epitope is a “conformationalepitope” composed of non-contiguous amino acids of a tumor antigen(tertiary structure). In some embodiments, the tumor antigen comprises arecombinant protein comprising both linear epitope(s) and conformationalepitope(s).

In some embodiments, the tumor antigen is encoded by a DNA or an mRNAmolecule. In some embodiments, the tumor antigen is encoded by a nucleicacid of a microbial vector, or said another way, the cancer vaccinecomprises a microbial vector. In some embodiments, the microbial vectoris a live, attenuated microbial vector. In one embodiment, the live,attenuated microbial vector is TICE® BCG, a live culture preparation ofthe Bacillus of Calmette and Guerin (BCG) strain of Mycobacterium bovis,marketed by Organon USA, Inc., (Roseland, N.J.). TICE® BCG is FederalDrug Administration (FDA)-approved for intravesical use uponreconstitution with sterile saline (e.g., pharmaceutically acceptableexcipient), and is indicated for the treatment and prophylaxis ofcarcinoma in situ of the urinary bladder, and for the prophylaxis ofprimary or recurrent stage Ta and/or T1 papillary tumors followingtransurethral resection.

In some embodiments, the microbial vector is a recombinant microbialvector, such as a recombinant viral vector or a recombinant bacterialvector. Recombinant viral vectors suitable for use in the combinationtherapies of the present disclosure include, but are not limited to,retroviruses, lentiviruses, adenoviruses, adeno-associated viruses,poxviruses, and herpesviruses (Chulpanova et al., Biomedicines, 6: 94,2018). In some embodiments, the microbial vector is a recombinantbacterial vector. Recombinant bacterial vectors suitable for use in thecombination therapies of the present disclosure include, but are notlimited to, Clostridium (C. novyi), Listeria (e.g., L. monocytogenes),Pseudomonas (e.g., P. aeruginosa), and Salmonella (S. typhimurium)(Toussant et al., Expert Rev Vaccines, 12: 1139-1154, 2013).

In some embodiments, the cancer vaccine comprises an antigen presentingcell (APC), that has been contacted with a tumor antigen, such as asynthetic peptide or a recombinant protein. In some embodiments, the APCis transfected with a nucleic acid encoding a tumor antigen. In someembodiments, the APC is transfected with a nucleic acid encoding a acytokine. In some embodiments, the APCs comprises dendritic cells ormesenchymal stem cells. In one embodiment, the cancer vaccine isPROVENGE® (sipuleucel-T) marketed by Dendreon Corp. (Seattle, Wash.).PROVENGE® comprises Lactated Ringer's (e.g., pharmaceutically acceptableexcipient) and peripheral blood mononuclear cells (PBMC) that have beenactivated with a PAP-GM-CSF fusion protein consisting of prostatic acidphosphatase linked to granulocyte-macrophage colony-stimulating factor.PROVENGE® is Federal Drug Administration (FDA)-approved for intravenousinfusion for the treatment of asymptomatic or minimally symptomaticmetastatic prostate cancer.

In some embodiments, the cancer vaccine comprises a killed tumor cell.In some embodiments, the cancer vaccine comprises a tumor cell lysate.In some embodiments, the cancer vaccine comprises an APC that has beencontacted with a tumor cell lysate.

Adjuvants of the cancer vaccines suitable for use in the methods,medicaments and uses of the present disclosure include, but are notlimited to, adjuvants of FDA-approved licensed products. In particular,adjuvants of current FDA-approved licensed products comprise aluminumsalts, monophosphoryl lipid A, oil-in-water emulsions (e.g.,squalene-in-water emulsions MF59 or AS03), saponins, and CpGoligodeoxynucleotides.

Oncolytic viruses suitable for use in the methods, medicaments, and usesof the present disclosure include, but are not limited to, adenovirus,coxsackievirus, echovirus, fowlpox virus, herpes simplex virus, marabavirus, measles virus, myxoma virus, Newcastle disease virus, parvovirus,poliovirus, retrovirus, reovirus, Seneca Valley virus, Semiliki Forestvirus, vaccinia virus, and vesicular stomatitis virus (see, e.g.,Russell and Peng, Chin Clin Oncol, 7: 16, 2018; and Sivanandam et al.,Molecular Therapy Oncolytics, 13: 93-106).

In some embodiments, the oncolytic virus has not beengenetically-engineered (non-recombinant virus). In some embodiments, thenon-recombinant virus is an echovirus (e.g., Rigvir), a Newcastledisease virus, a parvovirus, a reovirus, or a Seneca Valley virus.

In some embodiments, the oncolytic virus is a recombinant virus that hasbeen genetically engineered to include one or more gene deletions, oneor more gene insertions, or one or more gene deletions and one or moregene insertions. In some embodiments, the recombinant virus has beengenetically engineered to alter host cell specificity and/or tumor cellcytotoxicity. In some embodiments, the recombinant oncolytic virus hasbeen genetically engineered by functional deletion of one or more viralgenes encoding proteins that suppress a response (e.g., an anti-viralresponse) of a host cell, and/or by insertion of one or more transgenesencoding proteins that promote a response (e.g., an anti-tumor response)of a host cell (see, e.g., Guo et al., Frontiers in Immunology, 8:Article 555, 2017; and Lin et al., Oncology Letters, 15: 4053-4060,2018). In some embodiments, the recombinant virus is further engineeredby insertion of a transgene encoding a detectable marker, such asfluorescent protein. Desirable anti-tumor responses include one or bothof innate immune response and adaptive immune response.

In some embodiments, the recombinant oncolytic virus is a recombinantherpes simplex virus (HSV), such as HSV type-1. In one embodiment, therecombinant oncolytic virus is IMLYGIC®, also known as talimogenelaherparepvec or T-VEC, marketed by Amgen Inc. (Thousand Oaks, Calif.).IMILYGIC® is a recombinant HSV-1 that includes functional deletions ofICP34.5 and ICP47 genes, and insertion of a nucleic acid encoding humangranulocyte macrophage colony-stimulating factor (GM-CSF). IMLYGIC® isFederal Drug Administration (FDA)-approved for local treatment byintralesional injection (intratumoral administration) of unresectablecutaneous, subcutaneous, and nodal lesions in patients with recurrentmelanoma. In addition, IMLYGIC® is European Medicines Agency(EMA)-approved for treatment of adults with unresectable melanomas thatis regionally or distantly metastatic (Stages IIIB, IIIC or IVM1a). Inparticular, the EMA-approved product is to be administered byintralesional injection (intratumoral administration) into cutaneous,subcutaneous, and/or nodal lesions that are visible, palpable ordetectable by ultrasound guidance.

In some embodiments, the recombinant oncolytic virus is a recombinantadenovirus, such as a serotype 5 adenovirus. In one embodiment, therecombinant adenovirus is Oncorine (H101), formerly known as Onyx-015.Oncorine is a serotype 5 adenovirus engineered by inactivation(functional deletion) of viral E1B-55k and viral E3 genes. Oncorine isapproved by the Chinese State Food and Drug Administration for treatinghead and neck cancer in combination with chemotherapy (anti-neoplasticagent therapy).

In some embodiments, the recombinant oncolytic virus is a recombinantpox virus. In some embodiments, the poxvirus is a vaccinia virus or afowlpox virus. In some embodiments, the vaccinia virus is ModifiedVaccinia Ankara. In some embodiments, the recombinant vaccinia virus hasbeen genetically engineered by functional deletion of one or more viralgenes encoding proteins that suppress a response (e.g., an anti-viralresponse) of a host cell, and/or by insertion of one or more transgenesencoding proteins that promote a response (e.g., an anti-tumor response)of a host cell (see, e.g., Guo et al., Journal of ImmunoTherapy ofCancer, 7: 6, 2019). In one embodiment, the recombinant vaccinia virusis Pexa-Vec, also known as pexastimogene devacirepvec and JX-594, whichis a vaccinia virus engineered by inactivation (functional deletion) ofthe viral thymidine kinase gene, and by insertion of transgenes encodinghuman GM-CSF and beta-galactosidase (Heo et al., Nat Med, 19: 329-336,2013). In another embodiment, the recombinant vaccinia virus comprisesfunctional deletion of viral thymidine kinase and vaccinia growth factorgenes, and insertion of a transgene encoding the chemokine, CXCL11 (see,e.g., Liu et al., Oncolmmunology, 5: 3, e1091554, 2016; and Liu et al.,Nature Communications, 8: 14754, 2017).

Further embodiments of the combination therapies of the presentdisclosure comprise at least one additional therapeutic agent. In someembodiments, the at least one additional therapeutic agent is selectedfrom the group consisting of an immune checkpoint inhibitor,chemotherapy (antineoplastic agent), radiation therapy, and combinationsthereof.

In some embodiments, the immune checkpoint inhibitor is an antagonist ofat least one inhibitory immune checkpoint molecule. In some embodiments,the at least one inhibitory immune checkpoint molecule is selected fromthe group consisting PD-1 (CD279), PD-L1 (CD274), and CTLA4 (CD152). Theimmune checkpoint inhibitor may be a therapeutic biological product. Forinstance the immune checkpoint inhibitor may comprise an antibody orantigen-binding fragment thereof. The antibody or fragment may be amonoclonal antibody (mAb), a human antibody, a humanized antibody, or achimeric antibody, and may include a human constant region. In someembodiments the human constant region is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and incertain embodiments, the human constant region is an IgG1 or IgG4constant region. In some embodiments, the antibody or fragment is abispecific antibody. In some embodiments, the antigen-binding fragmentcomprises one of the group consisting of Fab, Fab′-SH, F(ab′)2, scFv,and Fv fragments.

In some embodiments, the chemotherapy comprises at least oneantineoplastic agent (i.e., WHO ATC code L01). In some embodiments, theat least one antineoplastic agent is selected from the group consistingof a cytotoxic antibiotic, a plant alkaloid, an antimetabolite, andalkylating agent, an other antineoplastic agent, and combinationsthereof. As used in reference to chemotherapy, the antineoplastic agentis a “drug”, as opposed to a “therapeutic biological product”.

In some embodiments, the radiation therapy is external beam radiationtherapy. In other embodiments, the radiation therapy is internalradiation therapy. In some embodiments, the radiation therapy isablative radiation therapy.

As used herein the term “biosimilar” refers to a biological product thatis similar to but without clinically meaningful differences in safetyand effectiveness from a Federal Drug Administration (FDA)-approvedreference product. For instance, there may be differences between abiosimilar product and a reference product in clinically inactivecomponents (e.g., differences in excipients of the formulations, minordifferences in glycosylation, etc.). Clinically meaningfulcharacteristics can be assessed through pharmacokinetic andpharmacodynamic studies. In some embodiments, the biosimilar product isan interchangeable product as determined by the FDA. In someembodiments, the cancer vaccine is a biosimilar of an FDA-approvedproduct.

IV. COMPOSITIONS, FORMULATIONS AND ROUTES OF ADMINISTRATION

Pharmaceutical compositions of any of the compounds disclosed herein, ora salt thereof, or solvate thereof, are embraced by the presentdisclosure. Thus, the disclosure includes pharmaceutical compositionscomprising a Cbl-b inhibitor, wherein the Cbl-B inhibitor is a compoundof formula (I), (I-A)-(I-N), (II-A)-(II-L), (III-A)-(III-D),(IV-A)-(IV-C), (V-A)-(V-G), or (VI-A)-(VI-D), or any variation thereofdisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof, and a pharmaceutically acceptable excipient, suchas a pharmaceutically acceptable vehicle or pharmaceutically acceptablecarrier. In some embodiments, the compound is a compound selected fromCompound Nos. 1-45 in Table 1, or a pharmaceutically acceptable salt orsolvate thereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof. In some embodiments, the compound is a compoundselected from Compound Nos. 47-52 in Table 1, or a pharmaceuticallyacceptable salt or solvate thereof, or tautomers thereof, orstereoisomers or mixtures of stereoisomers thereof. In one aspect, thepharmaceutically acceptable salt is an acid addition salt, such as asalt formed with an inorganic or organic acid.

The compounds and compositions disclosed herein may be administered inany suitable form and by any suitable route that will provide sufficientlevels of the compounds for treatment of the disease or disorder. Insome embodiments, the Cbl-b inhibitor and/or the additional therapeuticagent are administered by enteral administration. In some embodiments,the enteral administration is oral administration. In other embodiments,the Cbl-b inhibitor and/or the additional therapeutic agent areadministered by parenteral administration. In some embodiments, theparenteral administration is intratumoral injection. In someembodiments, the parenteral administration is by a route selected fromthe group consisting of intravenous, intraperitoneal, and subcutaneous.

Suitable routes of administration include oral administration, enteraladministration, parenteral administration including subcutaneousinjection, intravenous injection, intraarterial injection, intramuscularinjection, intrasternal injection, intraperitoneal injection,intralesional injection, intraarticular injection, intratumoralinjection, or infusion techniques. The compounds and compositions alsocan be administered sublingually, by mucosal administration, by buccaladministration, subcutaneously, by spinal administration, by epiduraladministration, by administration to cerebral ventricles, by inhalation(e.g., as mists or sprays), nasal administration, vaginaladministration, rectal administration, topical administration, ortransdermal administration, or by sustained release or extended releasemechanisms. The compounds and compositions can be administered in unitdosage formulations containing conventional pharmaceutically acceptablecarriers, excipients, adjuvants, and vehicles as desired. The compoundsand compositions may be administered directly to a specific or affectedorgan or tissue. The compounds can be mixed with pharmaceuticallyacceptable carriers, excipients, adjuvants, and vehicles to formcompositions appropriate for the desired route of administration. Insome embodiments, the compounds can be mixed with one or both of anantigen and an adjuvant. In some embodiments, the antigen is a cancerantigen.

In certain embodiments disclosed herein, especially those embodimentswhere a formulation is used for injection or other parenteraladministration, including the routes listed herein, but also includingany other route of administration described herein (such as oral,enteric, gastric, etc.), the formulations and preparations used in themethods are sterile. Sterile pharmaceutical formulations are compoundedor manufactured according to pharmaceutical-grade sterilizationstandards (United States Pharmacopeia Chapters 797, 1072, and 1211;California Business & Professions Code 4127.7; 16 California Code ofRegulations 1751, 21 Code of Federal Regulations 211) known to those ofskill in the art. A “sterile” formulation is aseptic, or free oressentially free from all living microorganisms and their spores.Examples of methods of sterilization of pharmaceutical formulationsinclude, but are not limited to, sterile filtration through sterilefiltration membranes, exposure to radiation such as gamma radiation, andheat sterilization.

Oral administration is advantageous due to its ease of implementationand patient compliance. If a patient has difficulty swallowing,introduction of medicine via feeding tube, feeding syringe, orgastrostomy can be employed in order to accomplish entericadministration. The active compound, and, if present, otherco-administered agents, can be enterally administered in any otherpharmaceutically acceptable excipient suitable for formulation foradministration via feeding tube, feeding syringe, or gastrostomy.

Intravenous administration also can be used advantageously, for deliveryof the compounds or compositions to the bloodstream as quickly aspossible and to circumvent the need for absorption from thegastrointestinal tract.

The compounds and compositions described for use herein can beadministered in solid form, in liquid form, in aerosol form, or in theform of tablets, pills, caplets, capsules (such as hard gelatin capsulesor soft elastic gelatin capsules), powder mixtures, granules,injectables, solutions, suppositories, enemas, colonic irrigations,emulsions, dispersions, food premixes, cachets, troches, lozenges, gums,ointments, cataplasms (poultices), pastes, powders, dressings, creams,patches, aerosols (e.g., nasal spray or inhalers), gels, suspensions(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsionsor water-in-oil liquid emulsions), elixirs, or in other forms suitablefor the route of administration. The compounds and compositions also canbe administered in liposome formulations. The compounds also can beadministered as prodrugs, where the prodrug undergoes transformation inthe treated subject to a therapeutically effective form.

In addition, pharmaceutical formulations may contain preservatives,solubilizers, stabilizers, re-wetting agents, emulgators, sweeteners,dyes, adjusters, and salts for the adjustment of osmotic pressure,buffers, coating agents, or antioxidants. Formulations comprising thecompound also may contain other substances that have valuabletherapeutic properties. Pharmaceutical formulations may be prepared byknown pharmaceutical methods. Additional formulations and methods ofadministration are known in the art. Suitable formulations can be found,e.g., in Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins, 21st ed. (2005), which is incorporated herein byreference.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to methods known inthe art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a parenterally acceptable diluentor solvent, for example, as a solution in propylene glycol. Among theacceptable vehicles and solvents that may be employed are water, saline,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono or di-glycerides. In addition, fatty acids suchas oleic acid may be used in the preparation of injectables.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, talc, or starch. Such dosage forms also may compriseadditional excipient substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms also may comprise buffering agents.Tablets and pills additionally can be prepared with enteric coatings.Acceptable excipients for gel capsules with a soft shell are, forinstance, plant oils, wax, fats, semisolid and liquid poly-ols, and soon.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions also may comprise additional agents, such as wettingagents, emulsifying and suspending agents, cyclodextrins, andsweetening, flavoring, and perfuming agents. Alternatively, the compoundalso may be administered in neat form if suitable.

The compounds and compositions also can be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono ormultilamellar hydrated liquid crystals that are dispersed in an aqueousmedium. Any physiologically acceptable and metabolizable lipid capableof forming liposomes can be used. The present compositions in liposomeform can contain stabilizers, preservatives, excipients, and the like,in addition to a compound as disclosed herein. Useful lipids include thephospholipids and phosphatidyl cholines (lecithins), both natural andsynthetic. Methods to form liposomes are known in the art. See, forexample, Gregoriadis, G. Ed., Liposome Technology, Third Edition:Liposome Technology: Liposome Preparation and Related Techniques, CRCPress, Boca Raton, Fla. (2006); and Prescott, Ed., Methods in CellBiology, Volume XIV, Academic Press, New York, N. W., p. 33 et seq(1976).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form can vary depending upon thepatient to whom the active ingredient is administered and the particularmode of administration. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the specific compound employed; the age,body weight, body area, body mass index (BMI), general health, sex, anddiet of the patient; the time of administration and route ofadministration used; the rate of excretion; and the drug combination, ifany, used. The compounds can be administered in a unit dosageformulation. The pharmaceutical unit dosage chosen is fabricated andadministered to provide sufficient concentration of drug in the patient,subject, or individual.

Although the compounds for use as described herein can be administeredas the sole active pharmaceutical agent, they also can be used incombination with one or more other agents. When additional active agentsare used in combination with the compounds for use as described herein,the additional active agents may generally be employed in therapeuticamounts as indicated in the Physicians' Desk Reference (PDR) 71stEdition (2017), which is incorporated herein by reference, or suchtherapeutically useful amounts as would be known to one of ordinaryskill in the art, or as are determined empirically for each patient.

Combinations of two or more of the compounds and compositions disclosedherein also can be used. The two or more compounds or compositions canbe mixed together shortly before administration and administeredtogether. The two or more compounds or compositions can be administeredsimultaneously, either by the same route of administration or bydifferent routes of administration. The two or more compounds orcompositions can be administered consecutively, either by the same routeof administration or by different routes of administration. In oneembodiment, a kit form can contain two or more compounds or compositionsas individual compounds or compositions, with printed or electronicinstructions for administration either as a mixture of compounds orcompositions, as separate compounds or compositions administeredsimultaneously, or as separate compounds or compositions administeredconsecutively. Where three or more compounds or compositions areadministered, they can be administered as a mixture of compounds orcompositions, as separate compounds or compositions administeredsimultaneously, as separate compounds or compositions administeredconsecutively, as separate compounds or compositions where two or moremay be administered simultaneously with the remainder administeredconsecutively before or after the simultaneous administration, or anyother possible combination of mixed administration, simultaneousadministration, and consecutive administration.

A compound as disclosed herein may in one aspect be in a purified formand compositions comprising a compound in purified forms are disclosedherein. Compositions comprising a compound as disclosed herein or a saltthereof are provided, such as compositions of substantially purecompounds. In some embodiments, a composition containing a compound asdisclosed herein or a salt thereof is in substantially pure form. In onevariation, “substantially pure” intends a composition that contains nomore than 35% impurity, wherein the impurity denotes a compound otherthan the compound (or compounds, if combinations of compounds are used)to be administered in the composition, or a salt or solvate of thecompound (or compounds, if combinations are used). The weight of anyadded vehicle, carrier, or excipient is excluded from such acalculation, and the added vehicle, carrier, or excipient is notconsidered as an impurity. For example, a composition of a substantiallypure compound selected from a compound of Table 1 refers to acomposition that contains no more than 35% impurity, wherein theimpurity denotes a compound other than the compound or a salt or solvatethereof. In one variation, a composition of substantially pure compoundor a salt or solvate thereof is provided wherein the compositioncontains no more than 25% impurity. In another variation, a compositionof substantially pure compound or a salt or solvate thereof is providedwherein the composition contains no more than 20% impurity. In stillanother variation, a composition of substantially pure compound or asalt or solvate thereof is provided wherein the composition contains nomore than 10% impurity. In a further variation, a composition ofsubstantially pure compound or a salt or solvate thereof is providedwherein the composition contains no more than 5% impurity. In anothervariation, a composition of substantially pure compound or a salt orsolvate thereof is provided wherein the composition contains no morethan 3% impurity. In still another variation, a composition ofsubstantially pure compound or a salt or solvate thereof is providedwherein the composition contains no more than 1% impurity. In a furthervariation, a composition of substantially pure compound or a salt orsolvate thereof is provided wherein the composition contains no morethan 0.5% impurity. In yet other variations, a composition ofsubstantially pure compound means that the composition contains no morethan 15%, no more than 10%, no more than 5%, no more than 3%, or no morethan 1% impurity. An impurity may be the compound in a stereochemicalform different from the desired stereochemical form. For instance, acomposition of substantially pure (S)-compound means that thecomposition contains no more than 15%, no more than 10%, no more than5%, no more than 3%, or no more than 1% of the (R)-form of the compound.Alternatively, as used herein, “enantiomeric excess (ee)” refers to adimensionless mol ratio describing the purity of chiral substances thatcontain, for example, a single stereogenic center. For instance, anenantiomeric excess of zero would indicate a racemic (e.g., 50:50mixture of enantiomers, or no excess of one enantiomer over the other).By way of further example, an enantiomeric excess of ninety-nine wouldindicate a nearly stereopure enantiomeric compound (i.e., large excessof one enantiomer over the other). The percentage enantiomeric excess, %ee=([(R)-compound]-[(S)-compound])/([(R)-compound]+[(S)-compound])×100,where the (R)-compound>(S)-compound; or %ee=([(S)-compound]-[(R)-compound])/([(S)-compound]+[(R)-compound])×100,where the (S)-compound>(R)-compound. Moreover, as used herein,“diastereomeric excess (de)” refers to a dimensionless mol ratiodescribing the purity of chiral substances that contain more than onestereogenic center. For example, a diastereomeric excess of zero wouldindicate an equimolar mixture of diastereoisomers. By way of furtherexample, diastereomeric excess of ninety-nine would indicate a nearlystereopure diastereomeric compound (i.e., large excess of onediastereomer over the other). Diastereomeric excess may be calculatedvia a similar method to ee. As would be appreciated by a person ofskill, de is usually reported as percent de (% de). % de may becalculated in a similar manner to % ee.

In some aspects, provided herein are compositions comprising a cellpopulation containing a modified immune cell such as those describedherein or produced by the methods disclosed herein. In some embodiments,the composition comprises a cell population containing a modified immunecell that has been in contact or is in contact with a Cbl-b inhibitordescribed herein or a composition thereof. In some embodiments, themodified immune cell has been or is in contact with an anti-CD3 antibodyalone. In some embodiments, the modified immune cell has been or is incontact with an anti-CD3 antibody in combination with an anti-CD28antibody. The provided compositions comprising a cell populationcontaining a modified immune cell described herein may further comprisea pharmaceutical acceptable excipient.

In some aspects, also provided herein is a cell culture compositioncomprising a cell population containing an immune cell and a Cbl-binhibitor described herein. In some embodiments, the immune cell is ahuman immune cell. In some embodiments, the immune cell is a cellselected from the group consisting of: a hematopoietic cell, amultipotent stem cell, a myeloid progenitor cell, a lymphoid progenitorcell, a T-cell, a B-cell, and a NK-cell. In some embodiments, the cellculture composition further comprises an anti-CD3 antibody. In someembodiments, the cell culture composition further comprises an anti-CD3antibody in combination with an anti-CD28 antibody. Methods forculturing cell compositions containing immune cells are well known inthe art and are contemplated herein.

A modified immune cell or compositions as described herein, e.g., acomposition comprising a cell population containing the modified immunecell or a pharmaceutical composition, can be provided in a suitablecontainer. Suitable containers include, for example, bottles, vials(e.g., dual chamber vials), syringes (e.g., single or dual chambersyringes), bags (e.g., an intravenous bag), and tubes (e.g., testtubes). The container may be formed from a variety of materials such asglass or plastic.

In some embodiments, a composition comprising a cell populationcontaining a modified immune cell as described herein (e.g., a cellculture composition) is provided in a culture vessel. A culture vesselas provided herein includes, but is not limited to, a tube (e.g., a testtube), a dish (e.g., a tissue culture dish), a bag, a multiwell plate(e.g., a 6-well tissue culture plate), and a flask (e.g., a cell cultureflask).

Also provided are the compositions as described herein for any usedescribed herein. In some embodiments, the compositions as describedherein are for preparation of a medicament for treating or preventing adisease or condition associated with Cbl-b activity. In someembodiments, the compositions as described herein are for preparation ofa medicament for treating cancer.

Pharmaceutical compositions of any of the compounds disclosed herein, ora salt or solvate thereof, for use in combination with a cancer vaccineare embraced by this disclosure. Thus, the disclosure includespharmaceutical compositions comprising a Cbl-b inhibitor for use incombination with a cancer vaccine, wherein the Cbl-b inhibitor is acompound of 1-719 (including “a” and “b” variants thereof) ofInternational Patent Appl. WO 2019/148005 or a compound of any ofFormula (I-A), Formula (I), Formula (II-A), Formula (II), Formula(III-A), Formula (III), or Formula (IV), or any variation thereofdisclosed therein, or a pharmaceutically acceptable salt or solvatethereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof.

In addition, pharmaceutical compositions of any of the compoundsdisclosed herein, or a salt or solvate thereof, for use in combinationwith an oncolytic virus are embraced by this disclosure. Thus, thedisclosure includes pharmaceutical compositions comprising a Cbl-binhibitor for use in combination with an oncolytic virus, wherein theCbl-b inhibitor is a compound of 1-719 (including “a” and “b” variantsthereof) of International Patent Appl. WO 2019/148005 or a compound ofany of Formula (I-A), Formula (I), Formula (II-A), Formula (II), Formula(III-A), Formula (III), or Formula (IV), or any variation thereofdisclosed therein, or a pharmaceutically acceptable salt or solvatethereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof. The compositions can further comprise apharmaceutically acceptable excipient, such as a pharmaceuticallyacceptable vehicle or pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition comprises both a smallmolecule Cbl-b inhibitor and a cancer vaccine. In some embodiments, thecompound is a compound selected from Compound Nos. 53-61 in Table 1A, ora pharmaceutically acceptable salt or solvate thereof, or tautomersthereof, or stereoisomers or mixtures of stereoisomers thereof. Inaddition, in some embodiments, the pharmaceutical composition comprisesboth a small molecule Cbl-b inhibitor and an oncolytic virus. In someembodiments, the compound is a compound selected from Compound Nos.53-61 in Table 1A, or a pharmaceutically acceptable salt or solvatethereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof. In some embodiments, the compound is a compoundselected from the Cbl-b inhibitors disclosed in the following patentapplications: compounds 1-719 (including “a” and “b” variants thereof)of International Patent Appl. WO 2019/148005 or a compound of any ofFormula (I-A), Formula (I), Formula (II-A), Formula (II), Formula(III-A), Formula (III), or Formula (IV) therein; compounds 1-45 of U.S.Patent Appl. No. 62/831,392 or a compound of Formula (I) therein;compounds 1-45 and 47-52 of U.S. Patent Appl. No. 62/866,909 or acompound of Formula (I) therein; compounds 1-45 and 47-52 of U.S. PatentAppl. No. 62/880,267 or a compound of Formula (I) therein; or anyvariation thereof, or a pharmaceutically acceptable salt or solvatethereof, or tautomers thereof, or stereoisomers or mixtures ofstereoisomers thereof.

In one aspect, the pharmaceutically acceptable salt is an acid additionsalt, such as a salt formed with an inorganic acid or an organic acid.

The compounds, vaccines, and compositions disclosed herein may beadministered in any suitable form and by any suitable route that willprovide sufficient levels of the compounds, vaccines, or compositionsfor treatment of the disease or disorder. In some embodiments, the Cbl-binhibitor and/or the cancer vaccine are administered by enteraladministration. The compounds, oncolytic viruses, and compositionsdisclosed herein may be administered in any suitable form and by anysuitable route that will provide sufficient levels of the compounds,oncolytic viruses, or compositions for treatment of the disease ordisorder. In some embodiments, the Cbl-b inhibitor and/or the oncolyticvirus are administered by enteral administration. In some embodiments,the enteral administration is oral administration. In other embodiments,the Cbl-b inhibitor and/or the cancer vaccine are administered byparenteral administration. In other embodiments, the Cbl-b inhibitorand/or the oncolytic virus are administered by parenteraladministration. In some embodiments, the parenteral administration isintratumoral injection. In some embodiments, the parenteraladministration is by a route selected from the group consisting ofintravenous, intraperitoneal, and subcutaneous.

Suitable routes of administration include oral administration, enteraladministration, parenteral administration including subcutaneousinjection, intravenous injection, intraarterial injection, intramuscularinjection, intrasternal injection, intraperitoneal injection,intralesional injection, intraarticular injection, intratumoralinjection, or infusion techniques. The compounds, vaccines, andcompositions also can be administered sublingually, by mucosaladministration, by buccal administration, subcutaneously, by spinaladministration, by epidural administration, by administration tocerebral ventricles, by inhalation (e.g., as mists or sprays), nasaladministration, vaginal administration, rectal administration, topicaladministration, or transdermal administration, or by sustained releaseor extended release mechanisms. The compounds, vaccines, andcompositions can be administered in unit dosage formulations containingconventional pharmaceutically acceptable carriers, excipients,adjuvants, and vehicles as desired. The compounds, vaccines, andcompositions may be administered directly to a specific or affectedorgan or tissue. The compounds and/or vaccines can be mixed withpharmaceutically acceptable carriers, excipients, adjuvants, andvehicles to form compositions appropriate for the desired route ofadministration. The compounds, oncolytic viruses, and compositions alsocan be administered sublingually, by mucosal administration, by buccaladministration, subcutaneously, by spinal administration, by epiduraladministration, by administration to cerebral ventricles, by inhalation(e.g., as mists or sprays), nasal administration, vaginaladministration, rectal administration, topical administration, ortransdermal administration, or by sustained release or extended releasemechanisms. The compounds, oncolytic viruses, and compositions can beadministered in unit dosage formulations containing conventionalpharmaceutically acceptable carriers, excipients, adjuvants, andvehicles as desired. The compounds, oncolytic viruses, and compositionsmay be administered directly to a specific or affected organ or tissue.The compounds and/or oncolytic viruses can be mixed withpharmaceutically acceptable carriers, excipients, adjuvants, andvehicles to form compositions appropriate for the desired route ofadministration.

In certain embodiments disclosed herein, especially those embodimentswhere a formulation is used for injection or other parenteraladministration, including the routes listed herein, but also includingany other route of administration described herein (such as oral,enteric, gastric, etc.), the formulations and preparations used in themethods are sterile, except for the presence of the presence of amicrobial vector in certain cancer vaccines. To prepare suchpreparations, all components of the formulations or preparations areprepared in a sterile manner before combining with the microbial vector.In certain embodiments disclosed herein, especially those embodimentswhere a formulation is used for injection or other parenteraladministration, including the routes listed herein, but also includingany other route of administration described herein (such as oral,enteric, gastric, etc.), the formulations and preparations used in themethods are sterile, except for the presence of the oncolytic virus. Toprepare such preparations, all components of the formulations orpreparations are prepared in a sterile manner before combining with theoncolytic virus. Sterile pharmaceutical formulations are compounded ormanufactured according to pharmaceutical-grade sterilization standards(United States Pharmacopeia Chapters 797, 1072, and 1211; CaliforniaBusiness & Professions Code 4127.7; 16 California Code of Regulations1751, 21 Code of Federal Regulations 211) known to those of skill in theart. A “sterile” formulation is aseptic, or free or essentially freefrom all living microorganisms and their spores. Examples of methods ofsterilization of pharmaceutical formulations include, but are notlimited to, sterile filtration through sterile filtration membranes,exposure to radiation such as gamma radiation, and heat sterilization.

Oral administration is advantageous due to its ease of implementationand patient compliance. If a patient has difficulty swallowing,introduction of medicine via feeding tube, feeding syringe, orgastrostomy can be employed in order to accomplish entericadministration. The active compound, vaccine, or composition, and, ifpresent, other co-administered agents, can be enterally administered inany other pharmaceutically acceptable excipient suitable for formulationfor administration via feeding tube, feeding syringe, or gastrostomy.The active compound, oncolytic virus, or composition, and, if present,other co-administered agents, can be enterally administered in any otherpharmaceutically acceptable excipient suitable for formulation foradministration via feeding tube, feeding syringe, or gastrostomy.

Intravenous administration also can be used advantageously, for deliveryof the compounds, vaccines, or compositions to the bloodstream asquickly as possible and to circumvent the need for absorption from thegastrointestinal tract. Intravenous administration also can be usedadvantageously, for delivery of the compounds, oncolytic viruses, orcompositions to the bloodstream as quickly as possible and to circumventthe need for absorption from the gastrointestinal tract.

The compounds, vaccines, and compositions described for use herein canbe administered in solid form, in liquid form, in aerosol form, or inthe form of tablets, pills, caplets, capsules (such as hard gelatincapsules or soft elastic gelatin capsules), powder mixtures, granules,injectables, solutions, suppositories, enemas, colonic irrigations,emulsions, dispersions, food premixes, cachets, troches, lozenges, gums,ointments, cataplasms (poultices), pastes, powders, dressings, creams,patches, aerosols (e.g., nasal spray or inhalers), gels, suspensions(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsionsor water-in-oil liquid emulsions), elixirs, or in other forms suitablefor the route of administration. The compounds, vaccines, andcompositions also can be administered in liposome formulations. Thecompounds, oncolytic viruses, and compositions described for use hereincan be administered in solid form, in liquid form, in aerosol form, orin the form of tablets, pills, caplets, capsules (such as hard gelatincapsules or soft elastic gelatin capsules), powder mixtures, granules,injectables, solutions, suppositories, enemas, colonic irrigations,emulsions, dispersions, food premixes, cachets, troches, lozenges, gums,ointments, cataplasms (poultices), pastes, powders, dressings, creams,patches, aerosols (e.g., nasal spray or inhalers), gels, suspensions(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsionsor water-in-oil liquid emulsions), elixirs, or in other forms suitablefor the route of administration. The compounds, oncolytic viruses, andcompositions also can be administered in liposome formulations. Thecompounds also can be administered as prodrugs, where the prodrugundergoes transformation in the treated subject to a therapeuticallyeffective form.

In addition, pharmaceutical formulations may contain preservatives,solubilizers, stabilizers, re-wetting agents, emulgators, sweeteners,dyes, adjusters, and salts for the adjustment of osmotic pressure,buffers, coating agents or antioxidants. Formulations comprising thecompound, vaccine, or composition also may contain other substances thathave valuable therapeutic properties. Formulations comprising thecompound, oncolytic virus, or composition also may contain othersubstances that have valuable therapeutic properties. Pharmaceuticalformulations may be prepared by known pharmaceutical methods. Additionalformulations and methods of administration are known in the art.Suitable formulations can be found, e.g., in Remington: The Science andPractice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2005),which is incorporated herein by reference.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions of the small molecule Cbl-b inhibitor, may beformulated according to methods known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a parenterally acceptable diluent or solvent, for example,as a solution in propylene glycol. Among the acceptable vehicles andsolvents that may be employed are water, saline, Ringer's solution, andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound, vaccine, or composition may be admixed with at leastone inert diluent such as sucrose, lactose, talc, or starch. In suchsolid dosage forms, the active compound, oncolytic virus, or compositionmay be admixed with at least one inert diluent such as sucrose, lactose,talc, or starch. Such dosage forms also may comprise additionalexcipient substances other than inert diluents, e.g., lubricating agentssuch as magnesium stearate. In the case of capsules, tablets, and pills,the dosage forms also may comprise buffering agents. Tablets and pillsadditionally can be prepared with enteric coatings. Acceptableexcipients for gel capsules with a soft shell are, for instance, plantoils, wax, fats, semisolid and liquid poly-ols, and so on.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions also may comprise additional agents, such as wettingagents, emulsifying and suspending agents, cyclodextrins, andsweetening, flavoring, and perfuming agents. Alternatively, the compoundalso may be administered in neat form if suitable.

The compounds, vaccines, and compositions also can be administered inthe form of liposomes. The compounds, oncolytic viruses, andcompositions also can be administered in the form of liposomes. As isknown in the art, liposomes are generally derived from phospholipids orother lipid substances. Liposomes are formed by mono or multilamellarhydrated liquid crystals that are dispersed in an aqueous medium. Anyphysiologically acceptable and metabolizable lipid capable of formingliposomes can be used. The present compositions in liposome form cancontain stabilizers, preservatives, excipients, and the like, inaddition to a compound or vaccine as disclosed herein. The presentcompositions in liposome form can contain stabilizers, preservatives,excipients, and the like, in addition to a compound or oncolytic virusas disclosed herein. Useful lipids include the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Gregoriadis,G. Ed., Liposome Technology, Third Edition: Liposome Technology:Liposome Preparation and Related Techniques, CRC Press, Boca Raton, Fla.(2006); and Prescott, Ed., Methods in Cell Biology, Volume XIV, AcademicPress, New York, N. W., p. 33 et seq (1976).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form can vary depending upon thepatient to whom the active ingredient is administered and the particularmode of administration. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the specific compound or vaccine employed;the age, body weight, body area, body mass index (BMI), general health,sex, and diet of the patient; the time of administration and route ofadministration used; the rate of excretion; and the drug combination, ifany, used. The compounds, vaccines, and compositions can be administeredin a unit dosage formulation. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the specific compound or oncolytic virusemployed; the age, body weight, body area, body mass index (BMI),general health, sex, and diet of the patient; the time of administrationand route of administration used; the rate of excretion; and the drugcombination, if any, used. The compounds, oncolytic viruses, andcompositions can be administered in a unit dosage formulation. Thepharmaceutical unit dosage chosen is fabricated and administered toprovide sufficient concentration of drug in the patient, subject, orindividual.

Although the compounds, vaccines, and compositions for use as describedherein can be administered as the sole active pharmaceutical agents,they also can be used in combination with one or more other agents. Whenadditional active agents are used in combination with the compounds,vaccines, or compositions for use as described herein, the additionalactive agents may generally be employed in therapeutic amounts asindicated in the Physicians' Desk Reference (PDR) 71st Edition (2017),which is incorporated herein by reference, or such therapeuticallyuseful amounts as would be known to one of ordinary skill in the art, oras are determined empirically for each patient. Although the compounds,oncolytic viruses, and compositions for use as described herein can beadministered as the sole active pharmaceutical agents, they also can beused in combination with one or more other agents. When additionalactive agents are used in combination with the compounds, oncolyticviruses, or compositions for use as described herein, the additionalactive agents may generally be employed in therapeutic amounts asindicated in the Physicians' Desk Reference (PDR) 71st Edition (2017),which is incorporated herein by reference, or such therapeuticallyuseful amounts as would be known to one of ordinary skill in the art, oras are determined empirically for each patient.

Combinations of two or more of the compounds, vaccines, and compositionsdisclosed herein also can be used. The two or more compounds, vaccines,or compositions can be mixed together shortly before administration andadministered together. The two or more compounds, vaccines, orcompositions can be administered simultaneously, either by the sameroute of administration or by different routes of administration. Thetwo or more compounds, vaccines, or compositions can be administeredconsecutively, either by the same route of administration or bydifferent routes of administration. In one embodiment, a kit form cancontain two or more compounds, vaccines, or compositions as individualcompounds, vaccines, or compositions, with printed or electronicinstructions for administration either as a mixture of compounds,vaccines, or compositions, as separate compounds, vaccines, orcompositions administered simultaneously, or as separate compounds,vaccines, or compositions administered consecutively. Where three ormore compounds, vaccines, or compositions are administered, they can beadministered as a mixture of compounds, vaccines, or compositions, asseparate compounds, vaccines, or compositions administeredsimultaneously, as separate compounds, vaccines, or compositionsadministered consecutively, as separate compounds, vaccines, orcompositions where two or more may be administered simultaneously withthe remainder administered consecutively before or after thesimultaneous administration, or any other possible combination of mixedadministration, simultaneous administration, and consecutiveadministration.

Combinations of two or more of the compounds, oncolytic viruses, andcompositions disclosed herein also can be used. The two or morecompounds, oncolytic viruses, or compositions can be mixed togethershortly before administration and administered together. The two or morecompounds, oncolytic viruses, or compositions can be administeredsimultaneously, either by the same route of administration or bydifferent routes of administration. The two or more compounds, oncolyticviruses, or compositions can be administered consecutively, either bythe same route of administration or by different routes ofadministration. In one embodiment, a kit form can contain two or morecompounds, oncolytic viruses, or compositions as individual compounds,oncolytic viruses, or compositions, with printed or electronicinstructions for administration either as a mixture of compounds,oncolytic viruses, or compositions, as separate compounds, oncolyticviruses, or compositions administered simultaneously, or as separatecompounds, oncolytic viruses, or compositions administeredconsecutively. Where three or more compounds, oncolytic viruses, orcompositions are administered, they can be administered as a mixture ofcompounds, oncolytic viruses, or compositions, as separate compounds,oncolytic viruses, or compositions administered simultaneously, asseparate compounds, oncolytic viruses, or compositions administeredconsecutively, as separate compounds, oncolytic viruses, or compositionswhere two or more may be administered simultaneously with the remainderadministered consecutively before or after the simultaneousadministration, or any other possible combination of mixedadministration, simultaneous administration, and consecutiveadministration.

A compound as disclosed herein for use in the pharmaceuticalcompositions and methods described herein may in one aspect be in apurified form and compositions comprising a compound in purified formsare disclosed herein. Compositions comprising a compound as disclosedherein or a salt thereof are provided, such as compositions ofsubstantially pure compounds. In some embodiments, a compositioncontaining a compound as disclosed herein or a salt thereof is insubstantially pure form. In one variation, “substantially pure” intendsa composition that contains no more than 35% impurity, wherein theimpurity denotes a compound other than the compound (or compounds, ifcombinations of compounds are used) to be administered in thecomposition, or a salt or solvate of the compound (or compounds, ifcombinations are used). The weight of any added vehicle, carrier, orexcipient is excluded from such a calculation, and the added vehicle,carrier, or excipient is not considered as an impurity. For example, acomposition of a substantially pure compound selected from a compound ofTable 1A refers to a composition that contains no more than 35%impurity, wherein the impurity denotes a compound other than thecompound or a salt or solvate thereof. In one variation, a compositionof substantially pure compound or a salt or solvate thereof is providedwherein the composition contains no more than 25% impurity. In anothervariation, a composition of substantially pure compound or a salt orsolvate thereof is provided wherein the composition contains no morethan 20% impurity. In still another variation, a composition ofsubstantially pure compound or a salt or solvate thereof is providedwherein the composition contains no more than 10% impurity. In a furthervariation, a composition of substantially pure compound or a salt orsolvate thereof is provided wherein the composition contains no morethan 5% impurity. In another variation, a composition of substantiallypure compound or a salt or solvate thereof is provided wherein thecomposition contains no more than 3% impurity. In still anothervariation, a composition of substantially pure compound or a salt orsolvate thereof is provided wherein the composition contains no morethan 1% impurity. In a further variation, a composition of substantiallypure compound or a salt or solvate thereof is provided wherein thecomposition contains no more than 0.5% impurity. In yet othervariations, a composition of substantially pure compound means that thecomposition contains no more than 15%, no more than 10%, no more than5%, no more than 3%, or no more than 1% impurity. An impurity may be thecompound in a stereochemical form different from the desiredstereochemical form. For instance, a composition of substantially pure(S)-compound means that the composition contains no more than 15%, nomore than 10%, no more than 5%, no more than 3%, or no more than 1% ofthe (R)-form of the compound.

Alternatively, as used herein, “enantiomeric excess (ee)” refers to adimensionless mol ratio describing the purity of chiral substances thatcontain, for example, a single stereogenic center. For instance, anenantiomeric excess of zero would indicate a racemic (e.g., 50:50mixture of enantiomers, or no excess of one enantiomer over the other).By way of further example, an enantiomeric excess of ninety-nine wouldindicate a nearly stereopure enantiomeric compound (i.e., large excessof one enantiomer over the other). The percentage enantiomeric excess, %ee=([(R)-compound]-[(S)-compound])/([(R)-compound]+[(S)-compound])×100,where the (R)-compound>(S)-compound; or %ee=([(S)-compound]-[(R)-compound])/([(S)-compound]+[(R)-compound])×100,where the (S)-compound>(R)-compound. Moreover, as used herein,“diastereomeric excess (de)” refers to a dimensionless mol ratiodescribing the purity of chiral substances that contain more than onestereogenic center. For example, a diastereomeric excess of zero wouldindicate an equimolar mixture of diastereoisomers. By way of furtherexample, diastereomeric excess of ninety-nine would indicate a nearlystereopure diastereomeric compound (i.e., large excess of onediastereomer over the other). Diastereomeric excess may be calculatedvia a similar method to ee. As would be appreciated by a person ofskill, de is usually reported as percent de (% de). % de may becalculated in a similar manner to % ee.

A compound, vaccine, or composition as disclosed herein can be providedin a suitable container. A compound, oncolytic virus, or composition asdisclosed herein can be provided in a suitable container. Suitablecontainers include, for example, bottles, vials (e.g., dual chambervials), syringes (e.g., single or dual chamber syringes), bags (e.g., anintravenous bag) and tubes (e.g., test tubes). The container may beformed from a variety of materials such as glass or plastic.

V. ARTICLES OF MANUFACTURE OR KITS

Also provided are articles of manufacture comprising any of thecompounds, pharmaceutical compositions, cells, modified immune cells,cell populations, cell compositions, cell cultures, or cell culturecompositions described herein. The articles of manufacture includesuitable containers or packaging materials for the compounds,pharmaceutical compositions, cells, modified immune cells, cellpopulations, cell compositions, cell cultures, or cell culturecompositions. Examples of a suitable container include, but are notlimited to, a bottle, a vial, a syringe, an intravenous bag, or a tube.For cells, modified immune cells, cell populations, cell compositions,cell cultures, or cell culture compositions, a suitable container can bea culture vessel, including, but not limited to, a tube, a dish, a bag,a multiwell plate, or a flask.

Also provided are kits comprising any of the compounds, pharmaceuticalcompositions, cells, modified immune cells, cell populations, cellcompositions, cell cultures, or cell culture compositions describedherein. The kits can contain the compounds, pharmaceutical compositions,cells, modified immune cells, cell populations, cell compositions, cellcultures, or cell culture compositions in suitable containers orpackaging materials, including, but not limited to, a bottle, a vial, asyringe, an intravenous bag, or a tube. The kits can contain cells,modified immune cells, cell populations, cell compositions, cellcultures, or cell culture compositions in a culture vessel, including,but not limited to, a tube, a dish, a bag, a multiwell plate, or aflask. The kits can comprise the compounds, pharmaceutical compositions,cells, modified immune cells, cell populations, cell compositions, cellcultures, or cell culture compositions for administration to anindividual in single-dose form or in multiple-dose form. The kits canfurther comprise instructions or a label for administering thecompounds, pharmaceutical compositions, cells, modified immune cells,cell populations, cell compositions, cell cultures, or cell culturecompositions to an individual according to any of the methods disclosedherein. The kits can further comprise equipment for administering thecompounds, pharmaceutical compositions, cells, modified immune cells,cell populations, cell compositions, cell cultures, or cell culturecompositions to an individual, including, but not limited to, needles,syringes, tubing, or intravenous bags. The kits can further compriseinstructions for producing any of the compounds, pharmaceuticalcompositions, cells, modified immune cells, cell populations, cellcompositions, cell cultures, or cell culture compositions disclosedherein.

Also provided are articles of manufacture comprising any of thecompounds, vaccines, or pharmaceutical compositions described herein.The articles of manufacture include suitable containers or packagingmaterials for the compounds, vaccines, or pharmaceutical compositions.Also provided are articles of manufacture comprising any of thecompounds, oncolytic viruses, or pharmaceutical compositions describedherein. The articles of manufacture include suitable containers orpackaging materials for the compounds, oncolytic viruses, orpharmaceutical compositions. Examples of a suitable container include,but are not limited to, a bottle, a vial, a syringe, an intravenous bagor a tube.

Also provided are kits comprising any of the compounds, vaccines, orpharmaceutical compositions described herein. The kits can contain thecompounds, vaccines, or pharmaceutical compositions in suitablecontainers or packaging materials, including, but not limited to, abottle, a vial, a syringe, an intravenous bag or a tube. The kits cancomprise the compounds, vaccines, or pharmaceutical compositions foradministration to an individual in single-dose form or in multiple-doseform. The kits can further comprise instructions or a label foradministering the compounds, vaccines, or pharmaceutical compositions toan individual according to any of the methods disclosed herein. The kitscan further comprise equipment for administering the compounds,vaccines, or pharmaceutical compositions to an individual, including,but not limited to, needles, syringes, tubing, or intravenous bags. Thekits can further comprise instructions for producing any of thecompounds, vaccines, or pharmaceutical compositions disclosed herein.

Also provided are kits comprising any of the compounds, oncolyticviruses, or pharmaceutical compositions described herein. The kits cancontain the compounds, oncolytic viruses, or pharmaceutical compositionsin suitable containers or packaging materials, including, but notlimited to, a bottle, a vial, a syringe, an intravenous bag or a tube.The kits can comprise the compounds, oncolytic viruses, orpharmaceutical compositions for administration to an individual insingle-dose form or in multiple-dose form. The kits can further compriseinstructions or a label for administering the compounds, oncolyticviruses, or pharmaceutical compositions to an individual according toany of the methods disclosed herein. The kits can further compriseequipment for administering the compounds, oncolytic viruses, orpharmaceutical compositions to an individual, including, but not limitedto, needles, syringes, tubing, or intravenous bags. The kits can furthercomprise instructions for producing any of the compounds, oncolyticviruses, or pharmaceutical compositions disclosed herein.

VI. EXEMPLARY EMBODIMENTS

The present disclosure is further described by the followingembodiments. The features of each of the embodiments are combinable withany of the other embodiments where appropriate and practical.

Embodiment 1. A compound of Formula (I-a):

-   -   or a tautomer thereof, or a pharmaceutically acceptable salt        thereof,    -   wherein:

is

-   -   X is CH or N;    -   Z¹ is CH or N;    -   Z² is CH or N;    -   R^(1a) and R^(1b) are independently H, C₁-C₆ alkyl, C₁-C₆        haloalkyl, or C₁-C₆ alkyl-OH;    -   R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkyl-OH,        C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl);    -   R^(2b) is H, halo, or C₁-C₆ alkyl;    -   or R^(2a) and R^(2b) are taken together with the carbon atom to        which they are attached to form a spiro 3- to 6-membered        heterocyclyl or a spiro C₃-C₆ cycloalkyl,        -   wherein at least one of the atoms of the spiro heterocyclyl            which is adjacent to the connecting piperidinyl ring is            carbon;    -   R^(3a) and R^(3b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form C₃-C₄ cycloalkyl;    -   R⁴ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH;

is

and Y is CR^(5a)R^(5b) or S;

-   -   or

is

and Y is a bond;

-   -   W is O or a bond;    -   R^(5a) and R^(5b) are independently H, halo, or C₁-C₆ alkyl;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form a C₃-C₆ cycloalkyl;    -   R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁷ is H, C₃-C₆ cycloalkyl, —NH-(3- to 6-membered heterocyclyl),        —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl), —O-(3- to 6-membered        heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆ cycloalkyl);    -   R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl;    -   R⁹ is H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and    -   R¹⁰ is —CF₃ or cyclopropyl.

Embodiment 2. The compound of embodiment 1, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

is

Embodiment 3. The compound of embodiment 2, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

Z¹ is CH.

Embodiment 4. The compound of embodiment 3, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

is

Embodiment 5. The compound of embodiment 2, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

Z¹ is N.

Embodiment 6. The compound of embodiment 5, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

is

Embodiment 7. The compound of embodiment 1, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

is

Embodiment 8. The compound of embodiment 7, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

Z² is CH.

Embodiment 9. The compound of embodiment 8, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

is

Embodiment 10. The compound of embodiment 7, or a tautomer thereof, or apharmaceutically acceptable salt thereof, wherein:

Z² is N.

Embodiment 11. The compound of embodiment 10, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

is

Embodiment 12. The compound of any one of embodiments 1-11, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

is

and Y is CR^(5a)R^(5b) or S.

Embodiment 13. The compound of embodiment 12, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

W is a bond.

Embodiment 14. The compound of embodiment 12, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

W is O.

Embodiment 15. The compound of any one of embodiments 1-11, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

is

and Y is a bond.

Embodiment 16. The compound of embodiment 15, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁸ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄ cycloalkyl.

Embodiment 17. The compound of embodiment 16, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁸ is —CH₃, —CF₃, or cyclopropyl.

Embodiment 18. The compound of any one of embodiments 1-17, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

X is CH.

Embodiment 19. The compound of any one of embodiments 1-17, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

X is N.

Embodiment 20. The compound of any one of embodiments 1-19, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

Y is a bond.

Embodiment 21. The compound of any one of embodiments 1-19, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

Y is CR^(5a)R^(5b).

Embodiment 22. The compound of embodiment 21, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

-   -   R^(5a) and R^(5b) are independently H, halo, or C₁-C₃ alkyl;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form a C₃-C₄ cycloalkyl.

Embodiment 23. The compound of embodiment 22, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

-   -   R^(5a) and R^(5b) are independently H, F, or —CH₃;    -   or R^(5a) and R^(5b) are taken together with the carbon atom to        which they are attached to form cyclopropyl.

Embodiment 24. The compound of embodiment 23, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R^(5a) and R^(5b) are independently H or F.

Embodiment 25. The compound of any one of embodiments 1-19, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

Y is S.

Embodiment 26. The compound of any one of embodiments 1-25, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R^(1a) and R^(1b) are independently H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, orC₁-C₃ alkyl-OH.

Embodiment 27. The compound of embodiment 26, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R^(1a) and R^(1b) are independently H, —CH₃, —CF₃, or —CH₂OH.

Embodiment 28. The compound of any one of embodiments 1-27, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R^(1b) is H.

Embodiment 29. The compound of any one of embodiments 1-28, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R^(2a) is —CN, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkyl-OH, C₁-C₃alkyl-CN, or —(C₁-C₃ alkylene)-O—(C₁-C₃ alkyl).

Embodiment 30. The compound of embodiment 29, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R^(2a) is —CN, —CH₃, —CF₃, —CH₂OH, —CH₂CN, or —CH₂—O—CH₃.

Embodiment 31. The compound of any one of embodiments 1-30, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R^(2b) is H, halo, or C₁-C₃ alkyl.

Embodiment 32. The compound of embodiment 31, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R^(2b) is H, F, or —CH₃.

Embodiment 33. The compound of any one of embodiments 1-28, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R^(2a) and R^(2b) are taken together with the carbon atom to which theyare attached to form a spiro 4- to 5-membered heterocyclyl or a spiroC₃-C₄ cycloalkyl.

Embodiment 34. The compound of embodiment 33, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R^(2a) and R^(2b) are taken together with the carbon atom to which theyare attached to form spiro cyclopropyl,

Embodiment 35. The compound of any one of embodiments 1-34, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

-   -   R^(3a) and R^(3b) are independently H, halo, or C₁-C₃ alkyl;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form C₃-C₄ cycloalkyl.

Embodiment 36. The compound of embodiment 35, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

-   -   R^(3a) and R^(3b) are independently H, F, or —CH₃;    -   or R^(3a) and R^(3b) are taken together with the carbon atom to        which they are attached to form cyclopropyl.

Embodiment 37. The compound of any one of embodiments 1-36, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R⁴ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃ alkyl-OH.

Embodiment 38. The compound of embodiment 37, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁴ is H, —CH₃, —CF₃, or —CH₂OH.

Embodiment 39. The compound of any one of embodiments 1-38, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R⁶ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄ cycloalkyl.

Embodiment 40. The compound of embodiment 39, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁶ is —CH₃, —CHF₂, or cyclopropyl.

Embodiment 41. The compound of any one of embodiments 1-40, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R⁷ is H, C₃-C₄ cycloalkyl, —NH(4- to 5-membered heterocyclyl), —NH(C₁-C₃alkyl), —NH(C₃-C₅ cycloalkyl), —O(C₁-C₃ alkyl), —O(4- to 5-memberedheterocyclyl), or —O(C₃-C₅ cycloalkyl).

Embodiment 42. The compound of embodiment 41, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁷ is H, cyclopropyl, —NH(CH₂CH₃), —NH(cyclopropyl), —OCH₂CH₃,—O(cyclopropyl),

Embodiment 43. The compound of any one of embodiments 1-42, or atautomer thereof, or a pharmaceutically acceptable salt thereof,wherein:

R⁹ is H, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃ alkyl-OH.

Embodiment 44. The compound of embodiment 43, or a tautomer thereof, ora pharmaceutically acceptable salt thereof, wherein:

R⁹ is H, —CH₃, —CF₃, or —CH₂OH.

Embodiment 45. A compound selected from the compounds in Table 1, or atautomer thereof, or a pharmaceutically acceptable salt thereof.

Embodiment 46. A pharmaceutical composition comprising the compound ofany one of embodiments 1-45, or a tautomer thereof, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.

Embodiment 47. A method of modulating activity of an immune cell, themethod comprising contacting the immune cell with an effective amount ofa Cbl-b inhibitor to modulate activity of the immune cell, wherein theCbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 48. The method of embodiment 47, wherein the immune cellcomprises a T-cell, a B-cell, or a natural killer (NK) cell.

Embodiment 49. The method of embodiment 47 or 48, wherein the immunecell is a tumor-infiltrating lymphocyte (TIL) isolated from a tumor of amammalian subject with cancer before the immune cell is contacted withthe Cbl-b inhibitor.

Embodiment 50. The method of any one of embodiments 47-49, furthercomprising isolating the immune cell from a tumor of a mammalian subjectwith cancer before the immune cell is contacted with the Cbl-binhibitor.

Embodiment 51. The method of any one of embodiments 47-50, wherein theimmune cell comprises a T-cell, and wherein modulating activity of theT-cell comprises one or more of increased T-cell activation, increasedT-cell proliferation, decreased T-cell exhaustion, and decreased T-celltolerance.

Embodiment 52. The method of embodiment 51, wherein increased T-cellactivation comprises increased production of a cytokine.

Embodiment 53. The method of embodiment 52, wherein the cytokinecomprises one or more selected from the group consisting of IL-2, IFN-γ,TNFα, and GM-CSF.

Embodiment 54. The method of embodiment 51 or 52, wherein increasedT-cell activation comprises increased cell surface expression of one ormore T-cell activation markers.

Embodiment 55. The method of embodiment 54, wherein the T-cellactivation markers comprise one or more selected from the groupconsisting of CD25, CD69, and CTLA4.

Embodiment 56. The method of any one of embodiments 51-55, wherein theT-cell has been or is in contact with an anti-CD3 antibody alone or incombination with an anti-CD28 antibody.

Embodiment 57. The method of any one of embodiments 51-55, furthercomprising culturing the immune cell with TL-2 alone or in combinationwith an anti-CD3 antibody and/or an anti-CD28 antibody.

Embodiment 58. The method of any one of embodiments 47-50, wherein theimmune cell comprises a NK-cell, and wherein modulating activity of anNK-cell comprises increased NK-cell activation.

Embodiment 59. The method of embodiment 58, wherein increased NK-cellactivation comprises increased production of a cytokine.

Embodiment 60. The method of embodiment 59, wherein the cytokinecomprises one or more selected from the group consisting of IFN-γ, TNFα,and MIP1P.

Embodiment 61. The method of any one of embodiments 47-60, wherein theimmune cell comprises a B-cell, and wherein modulating activity of aB-cell comprises increased B-cell activation, optionally whereinincreased B-cell activation comprises increased expression of CD69.

Embodiment 62. The method of any one of embodiments 47-61, wherein theimmune cell is a human immune cell.

Embodiment 63. The method of any one of embodiments 47-62, wherein theimmune cell comprises a recombinant chimeric receptor.

Embodiment 64. The method of embodiment 63, wherein the recombinantchimeric receptor is a chimeric antigen receptor.

Embodiment 65. A method of producing a modified immune cell, comprisingculturing a cell population containing an immune cell in the presence ofan effective amount of a Cbl-b inhibitor to modulate activity of theimmune cell, thereby producing the modified immune cell, wherein theCbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 66. The method of embodiment 65, further comprising culturingthe immune cell with an anti-CD3 antibody alone or in combination withan anti-CD28 antibody.

Embodiment 67. The method of embodiment 65, further comprising culturingof the immune cell with IL-2 alone or in combination with an anti-CD3antibody and/or an anti-CD28 antibody.

Embodiment 68. The method of any one of embodiments 65-67, furthercomprising recovering the modified immune cell.

Embodiment 69. The method of any one of embodiments 65-68, wherein theimmune cell is a tumor-infiltrating lymphocyte (TIL).

Embodiment 70. The method of any one of embodiments 65-68, wherein theimmune cell is a cell selected from the group consisting of: ahematopoietic cell, a multipotent stem cell, a myeloid progenitor cell,a lymphoid progenitor cell, a T-cell, a B-cell, and a NK-cell.

Embodiment 71. The method of any one of embodiments 65-68, wherein themodified immune cell is a cell selected from the group consisting of: ahematopoietic cell, a multipotent stem cell, a myeloid progenitor cell,a lymphoid progenitor cell, a T-cell, a B-cell, and a NK-cell.

Embodiment 72. The method of any one of embodiments 65-71, wherein theimmune cell is from an individual.

Embodiment 73. The method of any one of embodiments 65-72, wherein theimmune cell is a human immune cell.

Embodiment 74. The method of any one of embodiments 65-73, wherein theimmune cell or modified immune cell comprises a recombinant chimericreceptor.

Embodiment 75. The method of embodiment 74, wherein the recombinantchimeric receptor is a chimeric antigen receptor.

Embodiment 76. A modified immune cell produced by the method of any oneof embodiments 65-75.

Embodiment 77. A modified immune cell comprising a Cbl-b inhibitor,wherein the Cbl-b inhibitor is a compound of any one of embodiments1-45.

Embodiment 78. An isolated modified immune cell, wherein the immune cellhas been contacted or is in contact with a Cbl-b inhibitor, wherein theCbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 79. The modified immune cell of embodiment 78, wherein themodified immune cell is a T-cell, a B-cell or a NK-cell.

Embodiment 80. The modified immune cell of embodiment 78 or 79, whereinthe immune cell is a tumor-infiltrating lymphocyte (TIL) isolated from atumor of a mammalian subject with cancer before the immune cell iscontacted with the Cbl-b inhibitor.

Embodiment 81. The modified immune cell of any one of embodiments 78-80,wherein the modified immune cell is a T-cell, and wherein the T-cellexhibits one or more of increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and decreased T-celltolerance.

Embodiment 82. The modified immune cell of embodiment 81, whereinincreased T-cell activation comprises increased production of acytokine.

Embodiment 83. The modified immune cell of embodiment 82, wherein thecytokine comprises one or more selected from the group consisting ofIL-2, IFN-γ, TNFα, and GM-CSF.

Embodiment 84. The modified immune cell of any one of embodiments 81-83,wherein increased T-cell activation comprises increased cell surfaceexpression of one or more T-cell activation markers.

Embodiment 85. The modified immune cell of embodiment 84, wherein theT-cell activation markers comprise one or more selected from the groupconsisting of CD25, CD69, and CTLA4.

Embodiment 86. The modified immune cell of any one of embodiments 81-85,wherein the T-cell has been or is in contact with an anti-CD3 antibodyalone or in combination with an anti-CD28 antibody.

Embodiment 87. The modified immune cell of any one of embodiments 81-85,wherein the T-cell has been or is in contact with IL-2 alone or incombination with an anti-CD3 antibody and/or an anti-CD28 antibody.

Embodiment 88. The modified immune cell of any one of embodiments 78-80,wherein the modified immune cell is a NK-cell, and wherein the NK-cellexhibits increased NK-cell activation.

Embodiment 89. The modified immune cell of embodiment 88, whereinincreased NK-cell activation comprises increased production of acytokine.

Embodiment 90. The modified immune cell of embodiment 89, wherein thecytokine comprises one or more selected from the group consisting ofIFN-γ, TNFα, and MIP1P.

Embodiment 91. The modified immune cell of any one of embodiments 78-80,wherein the modified immune cell is a B-cell, and wherein the B-cellexhibits increased B-cell activation, optionally wherein increasedB-cell activation comprises increased expression of CD69.

Embodiment 92. The modified immune cell of any one of embodiments 78-91,wherein the modified immune cell is a human immune cell.

Embodiment 93. The modified immune cell of any one of embodiments 78-92,wherein the modified immune cell comprises a recombinant chimericreceptor.

Embodiment 94. The modified immune cell of embodiment 93, wherein therecombinant chimeric receptor is a chimeric antigen receptor.

Embodiment 95. A composition comprising a cell population containing themodified immune cell of any one of embodiments 76-94.

Embodiment 96. The composition of embodiment 95, further comprising apharmaceutically acceptable excipient.

Embodiment 97. The composition of embodiment 95, wherein the compositionis in a culture vessel.

Embodiment 98. The composition of embodiment 97, wherein the culturevessel is a tube, a dish, a bag, a multiwell plate, or a flask.

Embodiment 99. The composition of embodiment 95 or 96, wherein thecomposition is in a suitable container.

Embodiment 100. The composition of embodiment 99, wherein the suitablecontainer is a bottle, a vial, a syringe, an intravenous bag, or a tube.

Embodiment 101. A method of modulating the immune response, the methodcomprising administering an effective amount of the modified immune cellof any one of embodiments 76-94 or an effective amount of thecomposition of any one of embodiments 95-100 to an individual in needthereof.

Embodiment 102. The method of embodiment 101, wherein the individual hasa cancer.

Embodiment 103. A method of treating a cancer responsive to inhibitionof Cbl-b activity, the method comprising administering an effectiveamount of the modified immune cell of any one of embodiments 76-94 or aneffective amount of the composition of any one of embodiments 95-100 toan individual having the cancer responsive to inhibition of Cbl-bactivity.

Embodiment 104. The method of embodiment 102 or 103, wherein the canceris a hematologic cancer.

Embodiment 105. The method of embodiment 104, wherein the hematologiccancer is a lymphoma, a leukemia, or a myeloma.

Embodiment 106. The method of embodiment 102 or 103, wherein the canceris a non-hematologic cancer.

Embodiment 107. The method of embodiment 106, wherein thenon-hematologic cancer is a sarcoma or a carcinoma.

Embodiment 108. A method of inhibiting abnormal cell proliferation, themethod comprising administering an effective amount of the modifiedimmune cell of any one of embodiments 76-94 or an effective amount ofthe composition of any one of embodiments 95-100 to an individual inneed thereof.

Embodiment 109. The method of embodiment 108, wherein the abnormal cellproliferation is hyperplasia or cancer cell proliferation.

Embodiment 110. The method of embodiment 109, wherein the cancer cell isfrom a hematologic cancer.

Embodiment 111. The method of embodiment 110, wherein the hematologiccancer is a lymphoma, a leukemia, or a myeloma.

Embodiment 112. The method of embodiment 109, wherein the cancer cell isfrom a non-hematologic cancer.

Embodiment 113. The method of embodiment 112, wherein thenon-hematologic cancer is a sarcoma or a carcinoma.

Embodiment 114. A method of modulating the immune response, the methodcomprising administering an effective amount of a Cbl-b inhibitor to anindividual to modulate the immune response in the individual, whereinthe Cbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 115. A method of inhibiting Cbl-b activity, the methodcomprising administering an effective amount of a Cbl-b inhibitor to anindividual to inhibit Cbl-b activity in the individual, wherein theCbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 116. A method of treating a cancer responsive to inhibitionof Cbl-b activity, the method comprising administering an effectiveamount of a Cbl-b inhibitor to an individual to treat the cancerresponsive to inhibition of Cbl-b activity, wherein the Cbl-b inhibitoris a compound of any one of embodiments 1-45.

Embodiment 117. The method of embodiment 116, wherein the cancer is ahematologic cancer.

Embodiment 118. The method of embodiment 117, wherein the hematologiccancer is a lymphoma, a leukemia, or a myeloma.

Embodiment 119. The method of embodiment 116, wherein the cancer is anon-hematologic cancer.

Embodiment 120. The method of embodiment 119, wherein thenon-hematologic cancer is a sarcoma or a carcinoma.

Embodiment 121. The method of any one of embodiments 116-120, furthercomprising administering an effective amount of the modified immune cellof any one of embodiments 76-94 or an effective amount of thecomposition of any one of embodiments 95-100 to the individual to treatthe cancer.

Embodiment 122. A method of inhibiting abnormal cell proliferation, themethod comprising administering an effective amount of a Cbl-b inhibitorto an individual to inhibit abnormal cell proliferation in theindividual, wherein the Cbl-b inhibitor is a compound of any one ofembodiments 1-45.

Embodiment 123. The method of embodiment 122, wherein the abnormal cellproliferation is hyperplasia or cancer cell proliferation.

Embodiment 124. The method of embodiment 123, wherein the cancer cell isfrom a hematologic cancer.

Embodiment 125. The method of embodiment 124, wherein the hematologiccancer is a lymphoma, a leukemia, or a myeloma.

Embodiment 126. The method of embodiment 123, wherein the cancer cell isfrom a non-hematologic cancer.

Embodiment 127. The method of embodiment 126, wherein thenon-hematologic cancer is a sarcoma or a carcinoma.

Embodiment 128. The method of any one of embodiments 114-127, whereinthe individual has one or more of increased T-cell activation, increasedT-cell proliferation, decreased T-cell exhaustion, and decreased T-celltolerance after administration of the Cbl-b inhibitor.

Embodiment 129. The method of embodiment 128, wherein increased T-cellactivation comprises increased production of a cytokine.

Embodiment 130. The method of embodiment 129, wherein the cytokinecomprises one or more selected from the group consisting of IL-2, IFN-γ,TNFα, and GM-CSF.

Embodiment 131. The method of any one of embodiment 128-130, whereinincreased T-cell activation comprises increased cell surface expressionof one or more T-cell activation markers.

Embodiment 132. The method of embodiment 131, wherein the T-cellactivation markers comprise one or more selected from the groupconsisting of CD25, CD69, and CTLA4.

Embodiment 133. The method of any one of embodiments 114-132, whereinthe individual has increased NK-cell activation after administration ofthe Cbl-b inhibitor.

Embodiment 134. The method of embodiment 133, wherein increased NK-cellactivation comprises increased production of a cytokine.

Embodiment 135. The method of embodiment 134, wherein the cytokinecomprises one or more selected from the group consisting of IFN-γ, TNFα,and MIP1P.

Embodiment 136. The method of any one of embodiments 114-135, whereinthe individual has increased B-cell activation after administration ofthe Cbl-b inhibitor, optionally wherein increased B-cell activationcomprises increased expression of CD69.

Embodiment 137. A cell culture composition comprising a cell populationcontaining an immune cell and a Cbl-b inhibitor, wherein the Cbl-binhibitor is a compound of any one of embodiments 1-45.

Embodiment 138. The cell culture composition of embodiment 137, whereinthe immune cell is a cell selected from the group consisting of: ahematopoietic cell, a multipotent stem cell, a myeloid progenitor cell,a lymphoid progenitor cell, a T-cell, a B-cell, and a NK-cell.

Embodiment 139. The cell culture composition of embodiment 137 or 138,further comprising an anti-CD3 antibody alone or in combination with ananti-CD28 antibody.

Embodiment 140. The cell culture composition of any one of embodiments137-139, wherein the immune cell is an engineered immune cell comprisinga recombinant chimeric receptor.

Embodiment 141. The cell culture composition of embodiment 140, whereinthe recombinant chimeric receptor is a chimeric antigen receptor.

Embodiment 142. A pharmaceutical composition comprising a Cbl-binhibitor and one or both of an adjuvant and an antigen, wherein theCbl-b inhibitor is a compound of any one of embodiments 1-45.

Embodiment 143. The pharmaceutical composition of embodiment 142,wherein the antigen is a cancer antigen.

Embodiment 144. An article of manufacture comprising the modified immunecell of any one of embodiments 76-94, the composition of any one ofembodiments 95-100, the cell culture composition of any one ofembodiments 137-141, or the pharmaceutical composition of embodiment 46.

Embodiment 145. The article of manufacture of embodiment 144, whereinthe modified immune cell or cell culture composition is in a tube, adish, a bag, a multiwell plate, or a flask.

Embodiment 146. The article of manufacture of embodiment 144, whereinthe modified immune cell or pharmaceutical composition is in a bottle, avial, a syringe, an intravenous bag, or a tube.

Embodiment 147. A kit comprising the modified immune cell of any one ofembodiments 76-94 or the composition of any one of embodiments 95-100.

Embodiment 148. The kit of embodiment 147, wherein the modified immunecell is in a tube, a dish, a bag, a multiwell plate, or a flask.

Embodiment 149. The kit of embodiment 147, wherein the modified immunecell is in a bottle, a vial, a syringe, an intravenous bag, or a tube.

Embodiment 150. The kit of any one of embodiments 147-149, wherein thekit comprises instructions for administering the modified immune cell orcomposition to an individual according to the method of any one ofembodiments 101-113.

Embodiment 151. A kit comprising the pharmaceutical composition ofembodiment 46.

Embodiment 151. The kit of embodiment 151, wherein the kit comprisesinstructions for administering the pharmaceutical composition to anindividual according to the method of any one of embodiments 114-116.

Embodiment 152. A kit comprising the cell culture composition of any oneof embodiments 137-141.

Embodiment 153. The kit of embodiment 152, wherein the kit comprisesinstructions for producing a modified immune cell according to themethod of any one of embodiments 65-75.

Embodiment 154. A method for treating or preventing a disease orcondition associated with Cbl-b activity, the method comprisingadministering a Cbl-b inhibitor to an individual in need thereof,wherein the Cbl-b inhibitor is a compound of any one of embodiments1-45.

Embodiment 155. Use of a Cbl-b inhibitor in the manufacture of amedicament for treating or preventing a disease or condition associatedwith Cbl-b activity, wherein the Cbl-b inhibitor is a compound of anyone of embodiments 1-45.

Embodiment 156. Use of a Cbl-b inhibitor in the manufacture of amedicament for treating cancer, wherein the Cbl-b inhibitor is acompound of any one of embodiments 1-45.

Embodiment 157. A Cbl-b inhibitor for use in treating or preventing adisease or condition associated with Cbl-b activity, wherein the Cbl-binhibitor is a compound of any one of embodiments 1-45.

Embodiment 158. A Cbl-b inhibitor for use in treating cancer, whereinthe Cbl-b inhibitor is a compound of any one of embodiments 1-45.

The disclosure will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the disclosure. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

EXAMPLES

Examples A-O below describe syntheses of key intermediates used for thecompounds disclosed in Examples 1-52. General Work-up Procedure 1 refersto extraction of aqueous solutions with EtOAc 2-3 times; the combinedorganic extract was dried over sodium sulfate or anhydrous magnesiumsulfate, or was washed with brine or saturated aqueous ammonium chloridesolution before drying, filtration, and concentration under vacuum.

Preparative-scale chiral supercritical fluid chromatography (SFC) wasperformed using various CHIRALPAK columns, such as CHIRALPAK AS-H,CHIRALPAK AD-H, or CHIRALPAK IG, using solvent systems such as CO₂/MeOH,CO₂/EtOH, or CO₂/(MeOH+acetonitrile).

Preparative-scale chiral HPLC was performed using various CHIRALPAKcolumns, such as CHIRALPAK IA, CHIRAL ART Cellulose-SB, CHIRALPAK IF,and CHIRALPAK IG, using solvent systems such as hexane/methanol,hexane/ethanol, (hexane+dichloromethane)/ethanol, MTBE/methanol,MTBE/ethanol, and (hexane-8 mmol/L NH₃)/methanol, Mobile Phase B:ethanol and hexane/IPA.

Preparative-scale HPLC was performed using columns such as SunFire PrepC18 OBD, XBridge Prep OBD C₁₈, and XBridge Shield RP18 OBD, usingsolvent systems such as (water-0.1% formic acid)/acetonitrile, (water-10mmol/L NH₄HCO₃)/acetonitrile, or (water-10 mmol/L NH₄HCO₃)/acetonitrile.

Chromatography A refers to purification over silica gel, typically inpre-packed cartridges, eluting with mixtures of EtOAc in hexanes orpetroleum ether; Chromatography B refers to elution with mixtures ofMeOH in DCM; Chromatography C refers to use of C₁₈ reverse-phase silicagel, eluting with mixtures of acetonitrile in water; Chromatography Drefers to elution with mixtures of ethanol, EtOAc, and hexanes.Biological Examples are provided following Example 46. Compounds drawnwithout stereochemistry in Table 1 and/or Table 1A were tested asracemic or diasteromeric mixtures in the Biological Examples.

Abbreviations used in the Examples include the following: DAST:(Diethylamino)sulfur trifluoride; EDC:N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU:1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate; T3P: propylphosphonic anhydride; SPhos:2-Dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; XPhos:2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl; Xantphos:4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene; NBS:N-bromosuccinimide; NCS: N-chlorosuccinimide; BPO: benzoyl peroxide;THF: tetrahydrofuran; EtOAc: ethyl acetate; DCM: dichloromethane; MeOH:methanol; DCE: 1,2-dichloroethane; TEA: triethylamine; DIPEA:N,N-Diisopropylethylamine; and DMF: N,N-dimethylformamide.

Example A:3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)aniline (A)

Step 1: Synthesis of ethyl 2-(3-(3-nitrophenyl)oxetan-3-yl)acetate.Aqueous KOH (133.0 mL, 0.20 mol) was added to a suspension of[Rh(COD)Cl]₂ (3.2 g, 6.5 mmol) in dioxane (100 mL) and the mixture wasstirred for 30 min. 3-nitrophenylboronic acid (32.6 g, 0.20 mol) andsuccessively ethyl 2-(oxetan-3-ylidene)acetate (WO 2017/107907) (18.6 g,0.13 mol) in dioxane (40 mL) were added and the mixture was stirred atrt for 16 h under nitrogen. The reaction was quenched by the addition ofHCl (1 N) to pH=6˜7. The mixture was extracted with EtOAc (300 mL×3).The combined organic layers were washed with brine, dried, andconcentrated. The residue was purified by flash column chromatographywith 10-30% EtOAc in petroleum ether to afford the title compound (25.6g, 74%). MS (ESI) calculated for (C₁₃H₁₅NO₅) [M+H]⁺, 266.1; found,266.0.

Step 2: Synthesis of 2-(3-(3-nitrophenyl)oxetan-3-yl)acetohydrazide. Amixture of ethyl 2-(3-(3-nitrophenyl)oxetan-3-yl)acetate (20.0 g, 75.4mmol) in ethanol (100 mL) and hydrazine hydrate (20 mL) was stirred at80° C. for 16 h. The solvent was removed under vacuum. The residue wastriturated with EtOAc/petroleum ether (1/10) to afford the titlecompound (25.0 g, crude), which was used without purification. MS (ESI)calculated for (C₁₁H₁₃N₃O₄) [M+H]⁺, 252.1; found, 252.2.

Step 3: Synthesis ofN-methyl-2-(2-(3-(3-nitrophenyl)oxetan-3-yl)acetyl)hydrazinecarbothioamide.To a solution of 2-(3-(3-nitrophenyl)oxetan-3-yl)acetohydrazide (10.0 g,39.8 mmol) in THE (100 mL) was added isothiocyanatomethane (5.8 g, 79.7mmol). The solution was stirred at rt for 4 h. The solvent was removedunder vacuum. The residue was purified by Chromatography B to afford thetitle compound (10.0 g, 78%). MS (ESI) calculated for (C₁₃H₁₆N₄O₄S)[M+H]⁺, 325.1; found, 325.2.

Step 4: Synthesis of4-methyl-5-((3-(3-nitrophenyl)oxetan-3-yl)methyl)-4H-1,2,4-triazole-3-thiol.A mixture ofN-methyl-2-(2-(3-(3-nitrophenyl)oxetan-3-yl)acetyl)hydrazine-carbothioamide(10.0 g, 30.8 mmol) in sodium hydroxide (308 mL, 1 M) was stirred at rtfor 16 hours. The reaction was diluted with water. And then the pH valueof the solution was adjusted to 5 with HCl (1 N). The solids werecollected by filtration to afford the title compound (7.0 g), which wasused without purification. MS (ESI) calculated for (C₁₃H₁₄N₄O₃S) [M+1]⁺,307.1; found, 307.1.

Step 5: Synthesis of4-methyl-3-((3-(3-nitrophenyl)oxetan-3-yl)methyl)-4H-1,2,4-triazole. Toa solution of4-methyl-5-((3-(3-nitrophenyl)oxetan-3-yl)methyl)-4H-1,2,4-triazole-3-thiol(7.0 g, 22.8 mmol) in water (30 mL) was added NaNO₂ (15.8 g, 228.8mmol). This was followed by the addition of HNO₃ (228.8 mL, 1 M)dropwise with stirring at 0° C. and stirred for another 1 h at 0° C. Themixture was basified by saturated aqueous sodium bicarbonate solutionthen extracted with EtOAc (200 mL×3). The combined organic layers werewashed with brine, dried, and concentrated to afford the title compound(6 g, crude), which was used without purification. MS (ESI) calculatedfor (C₁₃H₁₄N₄O₃) [M+H]⁺, 275.1; found, 274.9.

Step 6: Synthesis of Example A. To a solution of4-methyl-3-((3-(3-nitrophenyl)oxetan-3-yl)methyl)-4H-1,2,4-triazole (10g, 36.5 mmol) in methanol (100 mL) was added Pd/C (dry, 4 g). Thesolution was stirred at rt for 16 h under hydrogen (2 atm). When thereaction was completed, the solids were filtered out. The filtrate wasconcentrated. The residue was purified by Chromatography C to afford thetitle compound (4.7 g, 53%). MS (ESI) calculated for (C₁₃H₁₆N₄O) [M+H]⁺,245.1; found, 245.0. ¹H NMR (300 MHz, DMSO-d₆) δ 8.19 (s, 1H), 6.92-6.87(m, 1H), 6.40 (J=8.1 Hz, 1H), 6.05 (s, 1H), 5.94 (J=7.5 Hz, 1H), 5.00(s, 2H), 4.90-4.84 (m, 2H), 4.79-4.74 (m, 2H), 3.38 (s, 2H), 2.83 (s,3H).

Example B:2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindoline-5-carbaldehyde(B)

Step 1: Synthesis of3-(methoxycarbonyl)-4-methyl-5-(trifluoromethyl)benzoic acid. To adegassed solution of methyl 5-bromo-2-methyl-3-(trifluoromethyl)benzoate(18.0 g, 60.8 mmol), oxalic acid (11.5 g, 91.2 mmol), acetic anhydride(9.3 g, 91.2 mmol) and N-ethyl-N-isopropylpropan-2-amine (11.8 g, 91.2mmol) in dimethylformamide (200 mL) were added Pd(OAc)₂ (1.4 g, 6.1mmol) and XantPhos (1.8 g, 3.0 mmol). The mixture was stirred at 100° C.for 16 h under nitrogen. The reaction was quenched by the addition ofHCl (1 M, 300 mL) to pH˜3. The aqueous solution was extracted with EtOAc(250 mL×3). The combined organic layers were dried, and concentrated.The residue was purified by Chromatography C to afford the titlecompound (7.5 g, 47%).

Step 2: Synthesis of4-(bromomethyl)-3-(methoxycarbonyl)-5-(trifluoromethyl)benzoic acid. Toa stirred solution of4-methyl-3-(methoxycarbonyl)-5-(trifluoromethyl)benzoic acid (8 g, 30.5mmol) and N-bromosuccinimide (NBS) (8.2 g, 46 mmol) in CCl₄ (160 mL) wasadded BPO (2.2 g, 9 mmol). The solution was stirred at 80° C. for 16 h.The mixture was concentrated. The crude product was purified byChromatography B to afford the title compound (8.0 g, 77%). MS (ESI)calculated for (C₁₁H₈BrF₃O₄) [M−H]⁻, 339.0; found, 339.1.

Step 3: Synthesis of methyl2-(bromomethyl)-5-(hydroxymethyl)-3-(trifluoro-methyl)benzoate. To astirred solution of4-(bromomethyl)-3-(methoxycarbonyl)-5-(trifluoro-methyl)benzoic acid(2.65 g, 7.77 mmol) in THE (30 mL) was added borane (19.4 mL, 19.4 mmol,1 M in THF). The solution was stirred at rt for 6 h. The reaction wasthen quenched by the addition of methanol (10 mL). The mixture wasconcentrated. The residue was purified by Chromatography A to afford thetitle compound (1.40 g, 84%).

Step 4: Synthesis of methyl2-(bromomethyl)-5-formyl-3-(trifluoromethyl)-benzoate. To a stirredsolution of methyl2-(bromomethyl)-5-(hydroxymethyl)-3-(trifluoro-methyl)benzoate (7.1 g,21.71 mmol) in EtOAc (70 mL) was added 2-iodoxybenzoic acid (9.1 g, 32.5mmol). The reaction was stirred at 70° C. for 3 h. The solids werefiltered out, and the filtrate was concentrated in vacuo. The residuewas purified by Chromatography A to afford the title compound (6.6 g,94%).

Step 5: Example B: A solution of Example A (11.3 g, 46.1 mmol) andmethyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (15.0 g,46.1 mmol) in acetonitrile (410 mL) and water (205 mL) was cooled to 0°C. before the addition of silver nitrate (10.2 g, 60.0 mmol) dissolvedin 58 mL water. The reaction was stirred for 40 h at room temperature atwhich point solid sodium bicarbonate was added until solution was pH-8.The mixture was then filtered through Celite, rinsing with acetonitrile(300 mL) followed by a DCM:EtOAc mixture (300 mL, 9:1). The organiclayer was separated and dried over sodium sulfate. The crude residue waspurified by Chromatography B. The oil obtained was azeotroped withtoluene (3×150 mL) to afford the title compound (10.5 g, 50%).

Example C:2,6-dichloro-4-[3-[(4-methyl-4H-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]pyridine

Step 1: Synthesis of 1,3-dimethyl2-(2,6-dichloropyridin-4-yl)propanedioate. To a mixture of2,4,6-trichloropyridine (150 g, 0.82 mol) in dimethylformamide (1.7 L)were added 1,3-dimethyl propanedioate (130 g, 0.99 mol) and K₂CO₃ (340g, 2.47 mol) at rt. The mixture was stirred at 70° C. for 16 h. Themixture was poured into water and extracted with EtOAc. The combinedorganic layers were washed with brine, dried, and concentrated undervacuum. The residue was purified by chromatography A to afford productas a semi-solid, which was triturated with EtOAc/petroleum ether (1/10).The solids were collected by filtration and dried under vacuum to affordthe title compound (75 g, 36%).

Step 2: Synthesis of 1,3-dimethyl2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane dioate. To amixture of 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)propanedioate (75 g,0.27 mol) and K₂CO₃ (111 g, 0.81 mol) in dimethylformamide (700 mL) wasadded 3-bromoprop-1-ene (65 g, 0.54 mol) dropwise at 0° C. The mixturewas stirred at 0° C. for 2 h. The reaction was quenched by the additionof water and extracted with EtOAc. The combined organic layers werewashed with brine, dried, and concentrated under vacuum. The residue waspurified by chromatography A to afford the title compound (74 g, 89%).

Step 3: Synthesis of2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane-1,3-diol. To amixture of 1,3-dimethyl2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propanedioate (30 g, 94mmol) in THF (310 mL) was added DIBAL-H (377 mL, 377 mmol, 1 M inhexane) dropwise at −40° C. The mixture was stirred at rt for 16 h. Thereaction was quenched by the addition of MeOH dropwise at −20° C. andstirred for 10 min below −10° C. The mixture was added to an aqueoussolution of Rochelle's salt at 10° C. and stirred for another 15 min.The mixture was extracted with EtOAc. The combined organic layers weredried. After filtration and evaporation, the residue was purified bychromatography B to afford the title compound (14 g, 56%).

Step 4: Synthesis of2,6-dichloro-4-[3-(prop-2-en-1-yl)oxetan-3-yl]pyridine. To a solution of2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane-1,3-diol (30 g,114 mmol) in toluene (500 mL) was added triphenylphosphene (60 g, 228mmol) at rt and stirred for 10 min. Zinc dimethyldithiocarbamate (Ziram,54 g, 176 mmol) and diethyl azodicarboxylate (39 g, 224 mmol) in toluene(60 mL) was added dropwise to the solution. The mixture was stirred atrt for 16 h. The solids were filtered off and the filtrate wasconcentrated under vacuum. The residue was purified by Chromatography Ato afford the title compound (20.1 g, 71%).

Step 5: Synthesis of3-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]propane-1,2-diol. To amixture of 2,6-dichloro-4-[3-(prop-2-en-1-yl)oxetan-3-yl]pyridine (20 g,81 mmol) and 4-methylmorpholine N-oxide (29 g, 245 mmol) in THE (500 mL)and water (125 mL) was added K₂OsO₄.2H₂O (603 mg, 1.64 mmol) at 0° C.The mixture was stirred at rt for 24 h. The reaction was quenched by theaddition of aqueous NaHSO₃. The mixture was stirred at rt for 10 min,and then extracted with DCM (3×). The combined organic layers werewashed with brine, dried, and concentrated under vacuum. The collectedorganic layer was used for the next step directly.

Step 6: Synthesis of2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetaldehyde. To a flask withsilica gel (306 g) was added the solution of NaIO₄ (35 g, 163 mmol) inwater (175 mL) and then stirred for 20 min. A solution of3-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]propane-1,2-diol in DCM (2.2L, crude) was added to the above solution at rt. After 3 h stirring, thesolids were filtered off and the filtrate was concentrated under vacuum.The residue was purified by chromatography A to afford the titlecompound (17.2 g, 85% over two steps).

Step 7: Synthesis of [3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]aceticacid. A mixture of2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetaldehyde (12 g, 48 mmol),t-BuOH (94 mL), NaClO₂ (15 g, 168 mmol) and NaH₂PO₄ (10 g, 84 mmol) in2-methyl-2-butene (70 mL) and water (70 mL) was stirred at rt for 16 h.The organic solvent was removed under vacuum. The mixture was acidifiedto pH˜3 by HCl (1 N) and extracted with EtOAc. The combined organiclayers were washed with brine, dried, and concentrated under vacuum.After filtration and evaporation, the residue was purified bychromatography B to afford the title compound (9.0 g, 70%).

Step 8: Synthesis of2-(2-(3-(2,6-dichloropyridin-4-yl)oxetan-3-yl)acetyl)-N-methylhydrazine-1-carbothioamide.To a mixture of [3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetic acid(12.0 g, 45.9 mmol) and 1-amino-3-methylthiourea (5.3 g, 50.5 mmol) indimethylformamide (100 mL) was added N,N-diisopropyl-ethylamine (17.8 g,137.9 mmol) and HATU (21.0 g, 55.1 mmol) at 0° C. The mixture wasstirred at rt for 16 h. The reaction was quenched by the addition ofwater and extracted with EtOAc. The combined organic layers were washedwith brine, dried, and concentrated under vacuum. The residue waspurified by chromatography A to afford the title compound (10.4 g, 65%).

Step 9: Synthesis of5-[[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]methyl]-4-methyl-4H-1,2,4-triazole-3-thiol.A mixture of2-(2-(3-(2,6-dichloropyridin-4-yl)oxetan-3-yl)acetyl)-N-methylhydrazine-1-carbothioamide(10.4 g, 29.8 mmol) in NaOH (1 M, 300 mL) was stirred at rt for 16 h.The mixture was acidified to pH˜3 by HCl (3 N). The mixture wasextracted with EtOAc and the combined organic layers were washed withbrine, dried, and concentrated under vacuum. Filtration andconcentration afforded the title compound (6.7 g, 68%), which was usedin next step directly without purification.

Step 10: Synthesis of2,6-dichloro-4-[3-[(4-methyl-4H-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]pyridine.To a mixture of5-[[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]methyl]-4-methyl-4H-1,2,4-triazole-3-thiol(6.7 g, 20.3 mmol) and NaNO₂ (8.4 g, 121.8 mmol) was added HNO₃ (1 N,122 mL) dropwise at 0° C. The mixture was stirred at rt for 16 h. Themixture was basified by saturated aqueous NaHCO₃ solution and extractedwith EtOAc. The combined organic layers were washed with brine, dried,and concentrated under vacuum. The residue was purified bychromatography C to afford the title compound (3.1 g, 51%).

Example D:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

To a stirring solution of Example G (5.00 g, 29.1 mmol) in DCM (300.00mL) was added triethylamine (24.2 mL, 175 mmol) and sodiumtriacetoxyborohydride (37.1 g, 175 mmol). The suspension was stirred for10 min and then cooled to 0° C. before methyl2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (9.47 g, 29.1 mmol)in 20 mL of DCM was added. The mixture was stirred at room temperaturefor about 12 h. The reaction was quenched with saturated ammoniumchloride followed by extraction with DCM (3×) and the combined organicswere dried over sodium sulfate, filtered, and concentrated. The cruderesidue was dissolved in methanol (100 mL) and then ammonia (7 N inmethanol, 100 mL) was added to the solution. The mixture was stirred atroom temperature for 12 hours. The crude was concentrated and thenpurified by Chromatography B to yield the title compound (7.10 g,70.0%); ¹H NMR (500 MHz, Chloroform-d) δ 8.05 (s, 1H), 7.83 (s, 1H),7.41 (s, 1H), 4.64 (s, 2H), 3.68 (s, 2H), 2.87-2.63 (m, 2H), 2.40 (td,J=11.5, 3.3 Hz, 1H), 2.22-1.94 (m, 4H), 1.09-0.98 (m, 3H). LCMS:C₁₆H₁₇F₅N₂O requires: 348, found: m/z=349 [M+H]⁺.

Example E and Example F:(R)-6-cyclopropyl-4-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)picolinicacid (E) and(S)-6-cyclopropyl-4-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)picolinicacid (F)

Step 1: Synthesis of ethyl 6-chloro-4-methylpicolinate. To a solution of6-chloro-4-methylpyridine-2-carboxylic acid (50.0 g, 58.48 mmol) in EtOH(300.0 mL) was added H₂SO₄ (4.00 mL) dropwise. The resulting mixture wasstirred at 70° C. for 16 h then concentrated under vacuum. The residuewas dissolved in EtOAc and washed sequentially with NaHCO₃ (sat. aq.)and water. The combined organic phases were dried, filtered, andconcentrated to afford the title compound (53.1 g, crude), which wasused in the next step without purification. MS (ESI) calculated for(C₉H₁₀ClNO₂) [M+H]⁺, 200.0, found, 200.0.

Step 2: Synthesis of ethyl 6-cyclopropyl-4-methylpicolinate. A degassedsolution of ethyl 6-chloro-4-methylpyridine-2-carboxylate (30.0 g, 0.15mol), cyclopropylboronic acid (25.0 g, 0.29 mol),tetrakis(triphenylphosphine)palladium (17.0 g, 14.71 mmol) and potassiumcarbonate (62.0 g, 0.45 mmol) in dioxane (450 mL) was stirred at 100° C.for 2 h under N₂. The reaction mixture was quenched by the addition ofwater and extracted with EtOAc. The organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum. Theresidue was purified by Chromatography A to provide the title compound(20.1 g, 64.8%). MS (ESI) calculated for (C₁₂H₁₅NO₂) [M+H]⁺, 206.1,found, 206.0.

Step 3: Synthesis of ethyl 6-cyclopropyl-4-formylpyridine-2-carboxylate.To a mixture of ethyl 6-cyclopropyl-4-methylpyridine-2-carboxylate (14.0g, 68.21 mmol), Ac₂O (31.5 g) in HOAc (120 mL) and was added SeO₂ (10.5g, 94.81 mmol). The resulting mixture was stirred at 120° C. for 16 h.The reaction mixture was cooled to room temperature and filtered. Thefiltrate was concentrated under vacuum. The crude residue was purifiedby Chromatography C to afford the title compound (9.8 g, 46.2%). MS(ESI) calculated for (C₁₂H₁₃NO₃) [M+H]⁺, 220.1, found, 220.0.

Step 4: Synthesis of ethyl6-cyclopropyl-4-[(4,4-difluoro-3-methylpiperidin-1-yl)methyl]pyridine-2-carboxylate.A mixture of ethyl 6-cyclopropyl-4-formylpyridine-2-carboxylate (500 mg,2.3 mmol), (4,4-difluoro-3-methylpiperidine hydrochloride (590 mg, 3.4mmol), triethylamine (480 μL, 3.4 mmol), sodium triacetoxyborohydride(1.0 g, 4.8 mmol) and DCM (9.0 mL) was heated in a sealed vial at 40-50°C. until LCMS indicated consumption of aldehyde. The mixture was dilutedwith MeOH and EtOAc, concentrated onto Celite, and purified byChromatography D to afford the title compound (567 mg, 74%).

Step 5: Synthesis of Example E and Example F. A mixture of6-cyclopropyl-4-[(4,4-difluoro-3-methylpiperidin-1-yl)methyl]pyridine-2-carboxylate(542 mg, 1.6 mmol) LiOH.H₂O (134 mg, 3.2 mmol), THE (4 mL) and water (2mL) was stirred vigorously for 2 h. The THF was removed under reducedpressure and the residue dried by lyophilization to afford racemic6-cyclopropyl-4-{[4,4-difluoro-3-methylpiperidin-1-yl]methyl}pyridine-2-carboxylicacid. The enantiomers were resolved by SFC over a (cellulosetris(3-chloro-4-methylphenylcarbamate) OZ column (Diacel) eluting withMeOH in CO₂ at 45° C. to afford 137 mg of Example E and 160 mg ofExample F.

Example G: (R)-4,4-difluoro-3-methylpiperidine hydrochloride

Step 1: Synthesis of 1-benzyl-4,4-difluoro-3-methylpiperidine. To amixture of 1-benzyl-3-methylpiperidin-4-one (200 g, 983 mmol) and HF(194 g, 9.68 mol, 176.00 mL, 100% purity) was added tetrafluoro-sulfane(240 g, 2.22 mol) in a stainless-steel autoclave at −78° C., then warmedto 15° C. and stirred for 16 h. The reaction mixture was added dropwiseinto saturated aq. Na₂CO₃ (2.0 L), extracted with EtOAc (200 mL×4),filtered, dried, and concentrated under vacuum to give the titlecompound (211 g, crude): ¹H NMR (400 MHz, CDCl₃) δ 7.24-7.23 (m, 5H),3.46 (s, 2H), 2.70-2.63 (m, 2H), 2.23-2.21 (m, 1H), 1.97-1.92 (m, 4H),0.91 (d, J 6.4 Hz, 3H).

Step 2: Synthesis of (R)-1-benzyl-4,4-difluoro-3-methylpiperidine. To asolution of 1-benzyl-4,4-difluoro-3-methylpiperidine (613 g, 2.42 mol)in isopropyl acetate (9 L) was added(2R,3R)-2,3-bis[(4-methylbenzoyl)oxy]butanedioic acid; hydrate (979 g,2.42 mol) at 30° C. The mixture was stirred at 30° C. for 2 h, thenheated to 70° C. for 2 h. The mixture was then cooled to 30° C. for 12h. The mixture was filtered, and the filtrate was adjusted to pH=8-9with 1 N aqueous sodium hydroxide solution then extracted with EtOAc(3×1500 mL), the combined organic layers were dried and concentratedunder vacuum to give the title compound (377 g, 1.67 mol, 62.3% yield,90.1% purity). This crude product (377 g, 1.67 mol) was dissolved inisopropyl acetate (5.6 L) and to the mixture was slowly added (2S,3S)-2,3-bis[(4-methylbenzoyl)oxy]butanedioic acid (646 g, 1.67 mol) at30° C. The mixture was stirred at 30° C. for 2 h, and then heated to 70°C. for 2 h. The mixture was cooled to 30° C. for 12 h. The precipitatewas collected by filtration and then dissolved in 1 N aqueous sodiumhydroxide to pH=8-9, and extracted with EtOAc (3×200 mL). The combinedorganic layers were dried, and concentrated under vacuum to give thetitle compound (125 g, 554.87 mmol, 33.1% yield, 98.6% purity) which wasused in the next step without further purification: ¹H NMR (400 MHz,DMSO-d₆) δ 7.34-7.25 (m, 5H), 3.51 (s, 2H), 2.69-2.66 (m, 2H), 2.22-2.21(m, 1H), 2.02-1.96 (m, 4H), 0.91 (d, J 6.8 Hz, 3H).

Step 3: Synthesis of (R)-4,4-difluoro-3-methylpiperidine hydrochloride.To a solution of (R)-1-benzyl-4,4-difluoro-3-methylpiperidine (115 g,510.48 mmol) in MeOH (2.5 L) was added Pd/C (40 g, 10% purity) under N₂.The suspension was degassed under vacuum and purged with H₂ three times.The reaction mixture stirred under H₂ (50 psi) at 25° C. for 12 h. Thereaction mixture was filtered, then cooled to 15° C. and HCl/dioxane (4M, 114.85 mL) as added, followed by stirring at 15° C. for 2 h. Themixture was concentrated to afford the title compound (50.11 g, 292mmol, 57.2% yield): ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H), 9.55 (s,1H), 3.51 (s, 2H), 3.43-3.32 (m, 2H), 3.32-3.29 (m, 1H), 2.71-2.68 (m,1H), 2.51-2.49 (m, 1H), 2.27-2.24 (m, 2H), 0.98 (d, J 6.8, 3H).

Example H:4-cyclopropyl-1-oxo-2H,3H-pyrrolo[3,4-c]pyridine-6-carbaldehyde

Step 1: Synthesis of 3-amino-6-chloro-2-iodopyridine-4-carboxylic acid.To a solution of 5-amino-2-chloropyridine-4-carboxylic acid (100.0 g,579.47 mmol) in DMF (1.4 L) was added N-iodosuccinimide (195.6 g, 869.21mmol) in portions at 0-10° C. After stirring for 30 min, the resultingmixture was heated to 80° C. and stirred for 16 h. The reaction mixturewas quenched by the addition of water followed by General Work-upProcedure 1 to afford the title compound (140.0 g, crude), which wasused in the next step without purification.

Step 2: Synthesis of methyl3-amino-6-chloro-2-iodopyridine-4-carboxylate. To a mixture of3-amino-6-chloro-2-iodopyridine-4-carboxylic acid (70.0 g, 234.80 mmol)in methanol (90.0 mL) and DCM (900.0 mL) was added TMSCHN₂ (176.1 mL, 2M in hexane) dropwise at 0° C. The resulting mixture was stirred at roomtemperature for 16 h. When the reaction was completed, the organicsolvents were removed under vacuum. The crude residue was trituratedwith 15% EtOAc in petroleum ether. The solids were collected byfiltration and dried to afford the title compound (65.0 g, crude), whichwas used in the next step without purification.

Step 3: Synthesis of methyl3-amino-6-chloro-2-cyclopropylpyridine-4-carboxylate. A degassed mixtureof methyl 3-amino-6-chloro-2-iodopyridine-4-carboxylate (45.0 g, 144.23mmol), cyclopropylboronic acid (49.6 g, 576.92 mmol), K₂CO₃ (59.7 g,432.69 mmol), Pd(dppf)C₁₂ (10.5 g, 14.42 mmol) in dioxane (1.5 L) wasstirred at 100° C. for 16 h under N₂ atmosphere. The solids werefiltered off and the filtrate was concentrated under vacuum. The cruderesidue was dissolved in EtOAc and water and General Work-up Procedure 1was followed before the residue was purified by Chromatography A toafford the title compound (22.1 g, 67% over three steps).

Steps 4 and 5: Synthesis of methyl6-chloro-2-cyclopropyl-3-iodopyridine-4-carboxylate. To a mixture ofmethyl 3-amino-6-chloro-2-cyclopropylpyridine-4-carboxylate (25.0 g,110.62 mmol) in DCM (1.5 L) were successively added t-BuONO (17.0 g,169.00 mmol) and BF₃.Et₂O (19.0 g, 264.70 mmol) dropwise at 0° C. underN₂ atmosphere. The resulting mixture was stirred at room temperature for2 h. The reaction mixture was quenched by the addition of hexane. Thesolids were collected by filtration and dried to afford6-chloro-2-cyclopropyl-4-(methoxycarbonyl)pyridine-3-diazoniumtetrafluoroborate (33.4 g), which was added to a mixture of KI (34.1 g,205.52 mmol) in water (250.0 mL) in portions at 50° C. The resultingmixture was stirred at 50° C. for 2 h. When the reaction was completed,General Work-up Procedure 1 was followed and the residue was purified byChromatography A to afford the title compound (23.0 g, 62% over twosteps).

Step 6: Synthesis of methyl6-chloro-2-cyclopropyl-3-methylpyridine-4-carboxylate. A degassedmixture of methyl 6-chloro-2-cyclopropyl-3-iodopyridine-4-carboxylate(23.0 g, 68.05 mmol), methylboronic acid (16.3 g, 272.20 mmol),Pd(dppf)Cl₂ (4.9 g, 6.81 mmol) and K₂CO₃ (28.2 g, 204.15 mmol) indioxane (500 mL) was stirred at 90° C. for 16 h under N₂ atmosphere. Thesolids were filtered off and the filtrate was concentrated under vacuum.The crude residue was dissolved in EtOAc and water and General Work-upProcedure 1 was followed before the crude was purified by ChromatographyA to afford the title compound (12.5 g, 83%).

Step 7: Synthesis of methyl3-(bromomethyl)-6-chloro-2-cyclopropylpyridine-4-carboxylate. A mixtureof methyl 6-chloro-2-cyclopropyl-3-methylpyridine-4-carboxylate (12.5 g,55.56 mmol) NBS (19.8 g, 111.11 mmol) and dibenzoyl peroxide (4.0 g,16.67 mmol) in CCl₄ (150.0 mL) was stirred at 80° C. for 16 h under N₂atmosphere. When the reaction was completed, the solids were filteredoff and the filtrate was concentrated under vacuum. The residue waspurified by Chromatography A to afford the title compound (7.8 g, 50%).

Step 8: Synthesis of6-chloro-4-cyclopropyl-2H,3H-pyrrolo[3,4-c]pyridin-1-one. A mixture ofmethyl 3-(bromomethyl)-6-chloro-2-cyclopropylpyridine-4-carboxylate (7.8g, 25.83 mmol) in NH₃ (7 M in MeOH, 80.0 mL) was stirred at roomtemperature for 16 h under N₂ atmosphere. The resulting mixture wasfiltered, the solids were collected and washed with methanol to affordthe title compound (5.0 g, 92.1%).

Step 9: Synthesis of4-cyclopropyl-6-ethenyl-2H,3H-pyrrolo[3,4-c]pyridin-1-one. A degassedmixture of 6-chloro-4-cyclopropyl-2H,3H-pyrrolo[3,4-c]pyridin-1-one (4.3g, 43.96 mmol), potassium vinyltrifluoroborate (11.8 g, 87.91 mmol),Pd(dppf)Cl₂.CH₂Cl₂ (3.6 g, 4.39 mmol) and Cs₂CO₃ (20.8 g, 131.88 mmol)in THF/water (v/v, 10/1, 220.0 mL) was stirred at 80° C. for 4 h underN₂ atmosphere. When the reaction was completed, the solvent was removedunder vacuum. The crude residue was dissolved with EtOAc and waterfollowed by General Work-up Procedure 1 and the resulting residue waspurified by Chromatography B to afford the title compound (3.5 g, 87%).

Step 10: Synthesis of4-cyclopropyl-1-oxo-2H,3H-pyrrolo[3,4-c]pyridine-6-carbaldehyde. To asolution of 4-cyclopropyl-6-ethenyl-2H,3H-pyrrolo[3,4-c]pyridin-1-one(3.5 g, 17.50 mmol) and 4-methylmorpholine N-oxide (6.1 g, 52.50 mmol)in THE (50 mL) and water (20 mL) was added K₂OsO₄.2H₂O (127.4 mg, 0.35mmol) at 0° C. The resulting mixture was stirred at room temperature for16 h. The reaction mixture was quenched by the addition of NaHSO₃ (10.2g) and stirred for 10 min at room temperature. Then to the mixture wasadded water (300.0 mL) and extracted with EtOAc. The aqueous layer wascollected to afford a crude solution of4-cyclopropyl-6-(1,2-dihydroxyethyl)-2H,3H-pyrrolo[3,4-c]pyridin-1-one(350 mL).

A three-necked flask was charged with silica gel (65.6 g) and a solutionof NaIO₄ (7.5 g, 35.00 mmol) in water (32.8 mL) was added dropwise whilevigorously stirring. After stirring for 20 min at room temperature, tothe resulting mixture was added the crude solution of4-cyclopropyl-6-(1,2-dihydroxyethyl)-2H,3H-pyrrolo[3,4-c]pyridin-1-one(350.0 mL in water) while vigorously stirring. The resulting mixture wasstirred at room temperature for 4 h. When the reaction was completed,the solids were filtered off and the filtrate was extracted with DCM.The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The residue was purified by Chromatography Ato afford the title compound (1.7 g, 48% over two steps): MS (ESI)calculated for (C₁₁H₁₀N₂O₂)[M+H]⁺, 203.1; found, 203.2. ¹H NMR (300 MHz,DMSO-d₆) δ 9.97 (s, 1H), 9.15 (s, 1H), 7.82 (s, 1H), 4.67 (s, 2H),2.29-2.21 (m, 1H), 1.22-1.14 (m, 4H).

Example I:(R)-4-cyclopropyl-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

A mixture of Example H (840 mg, 4.16 mmol), Example G (783 mg, 4.57mmol), and triethylamine (636 μL, 4.56 mmol) in DCM (17 mL) was stirredfor 10 min at rt before sodium triacetoxyborohydride (969 mg, 4.57 mmol)was added. After 1.5 h, additional sodium triacetoxyborohydride (177 mg)was added. The reaction was stirred overnight before being quenched withaq. sodium bicarbonate, followed by General Work-up Procedure 1 usingDCM. The crude material was purified by Chromatography B, followed byChromatography C to afford the title compound (626 mg, 47%): ¹H NMR (500MHz, DMSO-d₆) δ 8.90 (s, 1H), 7.41 (s, 1H), 4.52 (s, 2H), 3.68 (d, J=1.8Hz, 2H), 2.75 (t, J=14.5 Hz, 2H), 2.37-2.26 (m, 1H), 2.18-1.84 (m, 5H),1.09-0.96 (m, 4H), 0.92 (d, J=6.4 Hz, 3H); LCMS: C₁₇H₂₁F₂N₃O requires321.2, found 322.3 [M+H]⁺.

Example J:(S)-4-cyclopropyl-6-((3-methylpiperidin-1-yl)methyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

A solution4-cyclopropyl-1-oxo-2H,3H-pyrrolo[3,4-c]pyridine-6-carbaldehyde (1.0 g,4.95 mmol), (3S)-3-methylpiperidine hydrochloride (804 mg, 5.93 mmol),and triethylamine (0.69 mL, 4.56 mmol) in DCE (34 mL) was stirred for 15min at rt before sodium triacetoxyborohydride (1.36 g, 6.43 mmol) wasadded. The reaction was stirred overnight before being quenched with aq.sodium bicarbonate, followed by General Work-up Procedure 1 using DCM.The crude material was purified by Chromatography B to afford the titlecompound (1.1 g, 77%); LCMS: C₁₇H₂₃N₃O requires 285.2, found 286.2[M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 7.68 (s, 1H), 6.45 (s, 1H),4.57 (s, 2H), 3.69 (s, 2H), 2.83 (t, J=12.5 Hz, 2H), 2.05-1.98 (m, 1H),1.94 (ddd, J=12.9, 8.3, 4.7 Hz, 1H), 1.72 (d, J=9.0 Hz, 3H), 1.33-1.18(m, 3H), 1.06 (dt, J=8.1, 3.2 Hz, 2H), 0.86 (d, J=5.7 Hz, 5H).

Example K:2-chloro-6-ethoxy-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridine

A mixture of Example C (300 mg, 1.0 mmol) and EtONa (102 mg, 1.5 mmol)in ethanol (6 mL) was stirred at 80° C. for 16 h. The solvent wasremoved under vacuum and the crude was purified by chromatography C toafford the title compound (178 mg, 57%); ¹H NMR (300 MHz, DMSO-d₆) δ8.29 (s, 1H), 6.98 (d, J=1.2 Hz, 1H), 6.66 (d, J=1.2 Hz, 1H), 4.83-4.80(m, 4H), 4.24 (q, J=6.9 Hz, 2H), 3.58 (s, 2H), 3.35 (s, 3H), 1.29 (t,J=6.9 Hz, 3H); MS (ESI) calculated for C₁₄H₁₇C₁N₄O₂, 308; found, 309[M+H]⁺.

Example L:6-chloro-N-ethyl-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-amine

A mixture of Example C (300 mg, 1.0 mmol) in ethylamine (20 mL, 2 M inTHF) was stirred at 80° C. for 36 h in a sealed tube. The solvent wasremoved under vacuum and then the residue was purified by HPLC(acetonitrile in water with 0.1% trifluoroacetic acid) to afford thetitle compound (113 mg, 37%): ¹H NMR (300 MHz, DMSO-d₆) δ 8.29 (s, 1H),6.87 (t, J=5.4 Hz, 1H), 6.31 (d, J=1.2 Hz, 1H), 6.05 (d, J=1.2 Hz, 1H),4.82 (d, J=6.3 Hz, 2H), 4.74 (d, J=6.3 Hz, 2H), 3.47 (s, 2H), 3.42 (s,3H), 3.19-3.10 (m, 2H), 1.07 (t, J=7.2 Hz, 3H); MS (ESI) calculated forC₁₄H₁₈ClN₅O, 307; found, 308 [M+H]⁺.

Example M:3-((3-(3-bromophenyl)oxetan-3-yl)methyl)-4-(difluoromethyl)-4H-1,2,4-triazole

Step 1: Synthesis of [3-(3-bromophenyl)oxetan-3-yl]acetic acid. To asolution of ethyl 2-[3-(3-bromophenyl)oxetan-3-yl]acetate (WO2009/073300) (15.0 g, 50.3 mmol) in THE (150 mL) and water (120 mL) wasadded LiOH H₂O (4.2 g, 100.3 mmol) at 0° C. The mixture was stirred atroom temperature for 3 h. The organic solvent was removed under vacuumand then the residue was diluted with water and acidified to pH˜3 by HCl(1 N). The mixture was extracted with EtOAc three times. The combinedorganic layers were washed with brine, dried, and concentrated undervacuum to afford the title compound (12.0 g, crude). The crude productwas used for the next step without purification.

Step 2: Synthesis of 2-[3-(3-bromophenyl)oxetan-3-yl]acetamide. To amixture of [3-(3-bromophenyl)oxetan-3-yl]acetic acid (12.0 g, 44.4mmol), NH₄Cl (9.5 g, 177.7 mmol), 1-hydroxybenzotriazole (8.4 g, 62.2mmol) and EDC HCl (11.0 g, 57.7 mmol) in DMF (140 mL) was added DIPEA(28.7 g, 222 mmol) at 0° C. The resulting mixture was stirred at roomtemperature for 16 h. Water was added, and the solution was extractedwith EtOAc. The combined organic layers were washed with brine, dried,and concentrated under vacuum. The residue was purified bychromatography B to afford the title compound (7.1 g, 59%).

Step 3: Synthesis of(E)-2-(3-(3-bromophenyl)oxetan-3-yl)-N-((dimethylamino)methylene)acetamide.A solution of 2-[3-(3-bromophenyl)oxetan-3-yl]acetamide (7.1 g, 26.3mmol) in DMF-DMA (45.0 mL) was stirred at 80° C. for 16 h. The reactionmixture was poured into a mixture of water and brine. The resultingmixture was extracted with EtOAc. The combined organic layers were driedover Na₂SO₄. After filtration and evaporation, the residue was purifiedChromatography C to afford the title compound (5.6 g, 65%).

Step 4: Synthesis of3-[[3-(3-bromophenyl)oxetan-3-yl]methyl]-4H-1,2,4-triazole. A mixture of(E)-2-(3-(3-bromophenyl)oxetan-3-yl)-N-((dimethylamino)methylene)acetamide(5.6 g, 17.2 mmol) in acetic acid (50 mL) and hydrazine (50 mL, 80%) wasstirred at 80° C. for 4 h. The reaction mixture was concentrated and theresidue purified chromatography to afford the title compound (1.5 g,30%).

Step 5: Synthesis of3-[[3-(3-bromophenyl)oxetan-3-yl]methyl]-4-(difluoromethyl)-1,2,4-triazole.To a solution of3-[[3-(3-bromophenyl)oxetan-3-yl]methyl]-4H-1,2,4-triazole (1.5 g, 5.1mmol) in dimethylformamide (10 mL) was added NaH (2.1 g, 52.2 mmol, 60%purity) in portions at 0° C. and stirred for 30 min at room temperature.Then the reaction mixture was stirred at 28° C. for 16 h under CHF₂Cl(gas). The reaction mixture was purified directly by reverse phase flashcolumn chromatography with acetonitrile in water to afford 1.5 g ofproduct. The product was repurified by HPLC with acetonitrile in waterto afford the title compound (478 mg, 27%).

Example N:(S)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

To a stirring solution of (3S)-3-methylpiperidine hydrochloride (4.0 g,29.4 mmol) in DCM (250 mL) was added triethylamine (25 mL, 176 mmol) andsodium bis(acetyloxy)boranuidyl acetate (37.5 g, 176.9 mmol). Thesuspension was stirred for 10 min and then cooled to 0° C. Methyl2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (9.6 g, 29.4 mmol)in 20 mL of DCM was added. The mixture was allowed to stir at roomtemperature for about 12 h. The reaction was quenched with saturatedammonium chloride and extracted with DCM. The organic layer was dried,filtered, and concentrated. The crude was dissolved in methanol (100 mL)and then ammonia (7 N in methanol, 100 mL) was added to the solution.The mixture was stirred at room temperature for 12 hours. The crude wasconcentrated and then purified by Chromatography B to give6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one(4.1 g, 45%): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.18 (s, 1H), 8.14 (s,1H), 7.16 (s, 1H), 4.58 (s, 2H), 4.04 (s, 2H), 3.14-2.98 (m, 3H), 2.43(s, 1H), 2.18-2.10 (m, 1H), 2.04-1.73 (m, 2H), 1.34 (t, J=7.3 Hz, 1H),1.05 (qd, J=13.9, 12.9, 4.4 Hz, 1H), 0.89 (d, J=6.6 Hz, 3H); LCMS:C₁₆H₁₉F₃N₂O requires: 312, found: m/z=313 [M+H]⁺.

Example O:6-acetyl-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

Step 1:6-chloro-4-cyclopropyl-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one. Adegassed solution of 4,6-dichloro-2H,3H-pyrrolo[3,4-c]pyridin-1-one(2.03 g, 9.98 mmol), Pd(dppf)Cl₂.DCM complex (1.63 g, 2.00 mmol),2-cyclopropyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4.02 g, 24.0mmol) and K₂CO₃ (6.90 g, 49.9 mmol) in dioxane (25.5 mL) and H₂O (5.39mL) was stirred at 80° C. for 16 h under nitrogen atmosphere. GeneralWork-up Procedure 1 followed by Chromatography B and Chromatography Cafforded the title compound (670 mg, 32%).

Step 2:6-chloro-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.A solution of6-chloro-4-cyclopropyl-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one (1.91g, 9.15 mmol), potassium phosphate tribasic (5.83 g, 27.4 mmol),3-{[3-(3-bromophenyl)oxetan-3-yl]methyl}-4-methyl-1,2,4-triazole (2.82g, 9.15 mmol), Xantphos (1.06 g, 1.83 mmol), and palladium acetate(0.206 g, 0.92 mmol) in dioxane (38.7 mL) was stirred at 105° C. for 3 hunder nitrogen atmosphere. General Work-up Procedure 1 (extracted with15% iPrOH in CHCl₃) followed by Chromatography B afforded the titlecompound (2.32 g, 58%). LCMS: C₂₃H₂₂ClN₅O₂ requires: 435, found: m/z=436[M+H]⁺.

Step 3: Synthesis of6-acetyl-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.A mixture of6-chloro-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(314 mg, 0.721 mmol), tributyl(1-ethoxyvinyl)stannane (289 μL, 0.864mmol) and bis(triphenylphosphine)palladium(II) dichloride (42.2 mg,0.0601 mmol) in dioxane was purged with nitrogen and heated to 100° C.overnight. Upon cooling, the reaction mixture was pre-absorbed ontosilica gel and purified using a gradient of methanol in dichloromethane(0 to 20%) to obtain4-cyclopropyl-6-(1-ethoxyvinyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(278 mg). The mixture was dissolved in methanol (3 mL) and 1 N HCl (3mL) and stirred for 1 hour. Neutralization with saturated sodiumbicarbonate solution followed by dichloromethane extraction three times,drying, filtration, and concentration gave6-acetyl-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(268 mg, 84% yield).

Example 1:(S)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

A mixture of Example B (230 mg, 0.50 mmol, 1 equiv),(3S)-3-methylpiperidine hydrochloride (171 mg, 1.26 mmol, 2.5 equiv),triethylamine (175 μL, 1.26 mmol, 2.5 equiv), sodiumtriacetoxyborohydride (213 mg, 1.0 mmol, 2 equiv) and DCM (3.0 mL) washeated in a sealed vial at 40-50° C. until LCMS indicated consumption ofaldehyde. The mixture was diluted with water and acetonitrile,concentrated onto Celite, and purified by Chromatography C to afford thetitle compound: ¹H NMR (500 MHz, DMSO-d₆) δ 8.18 (s, 1H), 7.97 (s, 1H),7.92 (s, 1H), 7.88 (dd, J=8.1, 2.1 Hz, 1H), 7.40 (t, J=2.0 Hz, 1H), 7.35(t, J=7.9 Hz, 1H), 6.76 (d, J=7.6 Hz, 1H), 5.10 (s, 2H), 4.96 (d, J=6.0Hz, 2H), 4.89 (d, J=6.1 Hz, 2H), 3.65 (s, 2H), 3.51 (s, 2H), 2.90 (s,3H), 2.71 (t, J=11.5 Hz, 2H), 1.93 (s, 1H), 1.71-1.54 (m, 4H), 1.48 (d,J=12.4 Hz, 1H), 0.92-0.84 (m, 1H), 0.82 (d, J=6.0 Hz, 3H); LCMS:C₂₉H₃₂F₃N₅O₂ requires: 539, found: m/z=540 [M+H]⁺.

Example 2:6-{[(3R,5S)-3,5-dimethylpiperidin-1-yl]methyl}-2-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)-4-(trifluoromethyl)-3H-isoindol-1-one

The reductive amination was carried out in a similar fashion as forExample 1, using Example B (200 mg, 0.44 mmol) andcis-3,5-dimethylpiperidine (100 mg, 0.88 mmol) as reactants to affordthe title compound (100 mg, 41%): ¹H NMR (500 MHz, DMSO-d₆) δ 8.18 (s,1H), 7.97 (s, 1H), 7.91 (s, 1H), 7.88 (dd, J=8.1, 2.2 Hz, 1H), 7.40 (t,J=2.0 Hz, 1H), 7.35 (t, J=8.0 Hz, 1H), 6.76 (dt, J=7.6, 1.2 Hz, 1H),5.10 (s, 2H), 4.96 (d, J=6.1 Hz, 2H), 4.89 (d, J=6.0 Hz, 2H), 3.65 (s,2H), 3.51 (s, 2H), 2.90 (s, 3H), 2.75 (dd, J=10.5, 3.3 Hz, 2H),1.74-1.57 (m, 3H), 1.51 (t, J=10.8 Hz, 2H), 0.80 (d, J=6.4 Hz, 6H), 0.51(q, J=11.7 Hz, 1H); LCMS: C₃₀H₃₄F₃N₅O₂ requires: 553, found: m/z=554[M+H]⁺.

Example 3:(R)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

The reductive amination was carried out in a similar fashion as forExample 1, using Example B (50 mg, 0.11 mmol) and(3R)-3-methylpiperidine hydrochloride (30 mg, 0.22 mmol) as reactants toafford the title compound (12 mg, 21%): ¹H NMR (500 MHz, DMSO-d₆) δ 8.18(s, 1H), 7.97 (s, 1H), 7.92 (s, 1H), 7.88 (dd, J=8.0, 2.2 Hz, 1H), 7.40(t, J=2.0 Hz, 1H), 7.35 (t, J=8.0 Hz, 1H), 6.76 (dt, J=7.7, 1.2 Hz, 1H),5.09 (s, 2H), 4.96 (d, J=6.0 Hz, 2H), 4.89 (d, J=6.1 Hz, 2H), 3.65 (s,2H), 3.51 (s, 2H), 2.90 (s, 3H), 2.71 (t, J=11.2 Hz, 2H), 1.99-1.88 (m,1H), 1.71-1.55 (m, 4H), 1.49 (t, J=12.2 Hz, 1H), 0.92-0.84 (m, 1H), 0.82(d, J=6.1 Hz, 3H); LCMS: C₂₉H₃₂F₃N₅O₂ requires: 539, found: m/z=540[M+H]⁺.

Example 4:2-(1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidin-3-yl)acetonitrile

The reductive amination was carried out in a similar fashion as forExample 1, using Example B (50 mg, 0.11 mmol) and2-(piperidin-4-yl)acetonitrile hydrochloride (52.8 mg, 0.33 mmol) asreactants to afford the title compound (9 mg, 14%): ¹H NMR (500 MHz,DMSO-d₆) δ 8.20 (s, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 7.89 (dd, J=8.0,2.2 Hz, 1H), 7.41 (t, J=2.0 Hz, 1H), 7.36 (t, J=7.9 Hz, 1H), 6.78 (d,J=7.6 Hz, 1H), 5.11 (s, 2H), 4.98 (d, J=6.0 Hz, 2H), 4.90 (d, J=6.0 Hz,2H), 3.70 (s, 2H), 3.52 (s, 2H), 2.91 (s, 3H), 2.84-2.66 (m, 3H), 2.07(d, J=17.7 Hz, 1H), 1.89 (s, 3H), 1.75 (d, J=11.9 Hz, 1H), 1.65 (s, 1H),1.51 (d, J=11.2 Hz, 1H), 1.11 (t, J=11.5 Hz, 1H); LCMS: C₃₀H₃₁F₃N₆O₂requires: 564, found: m/z=565 [M+H]⁺.

Example 5 and Example 6:(R)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one (5) and(S)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(6)

Step 1: Synthesis of6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The reductive amination was carried out in a similar fashion as forExample 1 using Example B (55 mg, 0.12 mmol) and3-fluoro-3-methylpiperidine hydrochloride (55 mg, 0.36 mmol) asreactants to afford the title compound (22 mg, 32%).

Step 2: Separation of(R)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one and(S)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one:6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-onewas separated using an IG column with CO₂ and methanol as mobile phaseto afford the title compounds.(R)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one (9.6 mg): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 7.89 (s,1H), 7.85-7.77 (m, 3H), 7.27 (t, J=8.0 Hz, 1H), 7.23 (t, J=2.0 Hz, 1H),6.68 (ddd, J=7.7, 1.8, 1.0 Hz, 1H), 4.94 (d, J=6.1 Hz, 2H), 4.92-4.84(m, 4H), 3.60 (d, J=2.0 Hz, 2H), 3.44 (s, 2H), 2.78 (s, 3H), 2.57-2.46(m, 2H), 2.25-2.16 (m, 2H), 1.73-1.66 (m, 2H), 1.52-1.44 (m, 2H), 1.24(d, J=21.7 Hz, 3H); LCMS: C₂₉H₃₁F₄N₅O₂ requires: 557, found: m/z=558[M+H]⁺.(S)-6-((3-fluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one (9.6 mg): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 7.89 (s,1H), 7.85-7.78 (m, 3H), 7.27 (t, J=8.0 Hz, 1H), 7.23 (t, J=2.0 Hz, 1H),6.68 (ddd, J=7.7, 1.8, 1.0 Hz, 1H), 4.94 (d, J=6.1 Hz, 2H), 4.93-4.84(m, 4H), 3.60 (d, J=2.0 Hz, 2H), 3.44 (s, 2H), 2.78 (s, 3H), 2.57-2.48(m, 2H), 2.25-2.17 (m, 2H), 1.73-1.66 (m, 2H), 1.52-1.45 (m, 2H), 1.24(d, J=21.7 Hz, 3H); LCMS: C₂₉H₃₁F₄N₅O₂ requires: 557, found: m/z=558[M+H]⁺.

Example 7:6-((5-azaspiro[2.5]octan-5-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The reductive amination was carried out in a similar fashion as forExample 1 using Example B (150 mg, 0.33 mmol) and 5-azaspiro[2.5]octanehydrochloride (145 mg, 0.99 mmol) as reactants to afford the titlecompound (109 mg, 60%). ¹H NMR (500 MHz, Methanol-d₄) δ 8.17 (s, 1H),8.05-8.01 (m, 1H), 7.97 (s, 1H), 7.78-7.72 (m, 1H), 7.42-7.35 (m, 2H),6.73 (dt, J=7.7, 1.3 Hz, 1H), 5.09-5.04 (m, 6H), 3.68 (s, 2H), 3.65 (s,2H), 2.88 (s, 3H), 2.54 (s, 2H), 2.18 (s, 2H), 1.74 (p, J=5.7 Hz, 2H),1.35 (s, 2H), 0.35-0.26 (m, 4H); LCMS: C₃₀H₃₂F₃N₅O₂ requires: 551,found: m/z=552 [M+H]⁺.

Example 8 and Example 9:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(8) and(S)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(9)

Step 1: Synthesis of6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The reductive amination was carried out in a similar fashion as forExample 1 using Example B (50 mg, 0.11 mmol) and4,4-difluoro-3-methylpiperidine hydrochloride (56 mg, 0.33 mmol) asreactants to afford the title compound (23 mg, 36%).

Step 2: Separation of(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-oneand(S)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one:6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-onewas separated using an IG column with CO₂ and methanol as mobile phaseto afford the title compounds:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(8 mg): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 7.91 (s, 1H), 7.85-7.77 (m,3H), 7.27 (t, J=8.0 Hz, 1H), 7.23 (t, J=2.0 Hz, 1H), 6.68 (ddd, J=7.7,1.8, 1.0 Hz, 1H), 4.94 (d, J=6.1 Hz, 2H), 4.91-4.84 (m, 4H), 3.63 (s,2H), 3.44 (s, 2H), 2.75 (s, 3H), 2.73-2.61 (m, 2H), 2.29 (t, J=10.8 Hz,1H), 2.03-1.91 (m, 4H), 0.89 (d, J=6.4 Hz, 3H); LCMS: C₂₉H₃₀F₅N₅O₂requires: 575, found: m/z=576 [M+H]⁺; and(S)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(9 mg): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 7.91 (s, 1H), 7.86-7.78 (m,3H), 7.27 (t, J=8.0 Hz, 1H), 7.23 (t, J=2.0 Hz, 1H), 6.68 (ddd, J=7.7,1.8, 1.0 Hz, 1H), 4.94 (d, J=6.2 Hz, 2H), 4.93-4.84 (m, 4H), 3.63 (s,2H), 3.44 (s, 2H), 2.75 (s, 3H), 2.76-2.64 (m, 2H), 2.29 (t, J=10.8 Hz,1H), 2.06-1.94 (m, 4H), 0.89 (d, J=6.3 Hz, 3H); LCMS: C₂₉H₃₀F₅N₅O₂requires: 575, found: m/z=576 [M+H]⁺.

Example 10 and Example 11:(R)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile(10) and(S)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile(11)

Step 1: Synthesis of4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile.The reductive amination was carried out in a similar fashion as forExample 1 using Example B (180 mg, 0.39 mmol) and4,4-difluoropiperidine-3-carbonitrile hydrochloride (72 mg, 0.33 mmol)as reactants to afford the title compound (85 mg, 38%).

Step 2: Separation of(R)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrileand(S)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile.4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrilewas separated using an IG column with CO₂ and methanol as mobile phaseto afford the title compounds.(R)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile(1 mg). ¹H NMR (500 MHz, Methanol-d₄) δ 8.17 (s, 1H), 8.08 (s, 1H), 8.00(s, 1H), 7.76 (ddd, J=8.2, 2.2, 0.9 Hz, 1H), 7.43-7.37 (m, 2H), 6.74(ddd, J=7.6, 1.8, 0.9 Hz, 1H), 5.09-5.05 (m, 6H), 3.91-3.79 (m, 2H),3.65 (s, 2H), 3.64-3.51 (m, 1H), 2.89 (s, 3H), 2.88-2.75 (m, 2H), 2.70(s, 2H), 2.30-2.05 (m, 2H); LCMS: C₂₉H₂₇F₅N₆O₂ requires: 586, found:m/z=587 [M+H]⁺.(S)-4,4-difluoro-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile(1 mg). ¹H NMR (500 MHz, Methanol-d₄) δ 8.17 (s, 1H), 8.08 (s, 1H), 8.00(s, 1H), 7.76 (ddd, J=8.2, 2.2, 0.9 Hz, 1H), 7.43-7.38 (m, 2H), 6.74(ddd, J=7.6, 1.8, 0.9 Hz, 1H), 5.08-5.04 (m, 6H), 3.91-3.79 (m, 2H),3.65 (s, 2H), 3.64-3.51 (m, 1H), 2.89 (s, 3H), 2.88-2.75 (m, 2H), 2.70(s, 2H), 2.32-2.05 (m, 2H); LCMS: C₂₉H₂₇F₅N₆O₂ requires: 586, found:m/z=587 [M+H]⁺.

Example 12:6-((2-oxa-6-azaspiro[3.5]nonan-6-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The reductive amination was carried out in a similar fashion as forExample 1 using Example B (200 mg, 0.44 mmol) and2-oxa-6-azaspiro[3.5]nonane hydrochloride (143 mg, 0.88 mmol) asreactants to afford the title compound (143 mg, 58%). ¹H NMR (500 MHz,DMSO-d₆) δ 8.20 (s, 1H), 8.01 (s, 1H), 7.96 (s, 1H), 7.92-7.86 (m, 1H),7.42 (t, J=2.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 6.77 (dt, J=7.8, 1.1 Hz,1H), 5.12 (s, 2H), 4.98 (d, J=6.0 Hz, 2H), 4.90 (d, J=6.1 Hz, 2H), 4.27(d, J=5.8 Hz, 2H), 4.17 (d, J=5.8 Hz, 2H), 3.72 (s, 2H), 3.52 (s, 2H),2.91 (s, 3H), 2.36 (s, 2H), 1.66 (s, 2H), 1.49 (q, J=5.8 Hz, 2H); LCMS:C₃₀H₃₂F₃N₅O₃ requires: 567, found: m/z=568 [M+H]⁺.

Example 13 and Example 14:(R)-6-((10,10-difluoro-2-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(13) and(S)-6-((10,10-difluoro-2-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(14)

Step 1: Synthesis of6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The reductive amination was carried out in a similar fashion as forExample 1 using Example B (200 mg, 0.44 mmol) and10,10-difluoro-2-oxa-7-azaspiro[4.5]decane hydrochloride (187 mg, 0.88mmol) as reactants to afford the title compound (148 mg, 54%).

Step 2: Separation of(R)-6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-oneand(S)-6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-onewas separated using a Chiralpak IF column with CO₂ and methanol asmobile phase to afford the title compounds.(R)-6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(36 mg). ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.02 (s, 1H), 7.98(s, 1H), 7.89 (dd, J=8.1, 2.1 Hz, 1H), 7.41 (dd, J=2.0 Hz, 1H), 7.36(dd, J=7.9 Hz, 1H), 6.80-6.74 (m, 1H), 5.12 (s, 2H), 4.98 (d, J=6.0 Hz,2H), 4.90 (d, J=6.1 Hz, 2H), 3.77 (d, J=20.5 Hz, 3H), 3.67 (td, J=7.0,2.5 Hz, 3H), 3.60 (s, 1H), 3.52 (s, 2H), 2.91 (s, 3H), 2.49 (s, 2H),2.41 (s, 1H), 2.11-1.99 (m, 3H); LCMS: C₃₁H₃₂F₅N₅O₃ requires: 617,found: m/z=618 [M+H]⁺.(S)-6-((10,10-difluoro-1-oxa-7-azaspiro[4.5]decan-7-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(41 mg). ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.02 (s, 1H), 7.98(s, 1H), 7.89 (dd, J=8.1, 2.1 Hz, 1H), 7.41 (dd, J=2.0 Hz, 1H), 7.36(dd, J=7.9 Hz, 1H), 6.82-6.74 (m, 1H), 5.12 (s, 2H), 4.98 (d, J=6.0 Hz,2H), 4.90 (d, J=6.1 Hz, 2H), 3.77 (d, J=20.5 Hz, 3H), 3.67 (td, J=7.0,2.5 Hz, 3H), 3.60 (s, 1H), 3.52 (s, 2H), 2.91 (s, 3H), 2.49 (s, 2H),2.41 (s, 1H), 2.10-2.00 (m, 3H); LCMS: C₃₁H₃₂F₅N₅O₃ requires: 617,found: m/z=618 [M+H]⁺.

Example 15 and Example 16:(S)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-oneand (15) and(R)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(16)

Step 1:6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The reductive amination was carried out in a similar fashion as forExample 1 using Example B (280 mg, 0.61 mmol) and(4,4-difluoropiperidin-3-yl)methanol hydrochloride (230 mg, 1.23 mmol)as reactants to afford the title compound (43 mg, 12%).

Step 2: Separation of(R)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-oneand(S)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.6-((4,4-Difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-onewas separated using an IG column with CO₂ and methanol as mobile phaseto afford the title compounds.(R)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(7 mg). ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.02 (s, 1H), 7.97 (s,1H), 7.89 (dd, J=8.3, 2.2 Hz, 1H), 7.40 (dd, J=2.0 Hz, 1H), 7.36 (dd,J=8.0 Hz, 1H), 6.78 (dt, J=7.7, 1.2 Hz, 1H), 5.11 (s, 2H), 4.98 (d,J=6.0 Hz, 2H), 4.90 (d, J=6.1 Hz, 2H), 4.66 (dd, J=6.0, 4.7 Hz, 1H),3.88-3.68 (m, 3H), 3.52 (s, 2H), 2.91 (s, 3H), 2.78 (d, J=10.6 Hz, 1H),2.34 (s, 1H), 2.13 (d, J=23.7 Hz, 1H), 2.05-1.94 (m, 2H); LCMS:C₂₉H₃₀F₅N₅O₃ requires: 591, found: m/z=592 [M+H]⁺.(S)-6-((4,4-difluoro-3-(hydroxymethyl)piperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one(8 mg). ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.02 (s, 1H), 7.97 (s,1H), 7.89 (dd, J=8.3, 2.2 Hz, 1H), 7.40 (dd, J=2.0 Hz, 1H), 7.36 (dd,J=8.0 Hz, 1H), 6.78 (dt, J=7.7, 1.2 Hz, 1H), 5.11 (s, 2H), 4.98 (d,J=6.0 Hz, 2H), 4.90 (d, J=6.1 Hz, 2H), 4.66 (dd, J=6.0, 4.7 Hz, 1H),3.89-3.71 (m, 3H), 3.52 (s, 2H), 2.91 (s, 3H), 2.78 (d, J=10.6 Hz, 1H),2.34 (s, 1H), 2.13 (d, J=23.7 Hz, 2H), 2.04-1.96 (m, 2H); LCMS:C₂₉H₃₀F₅N₅O₃ requires: 591, found: m/z=592 [M+H]⁺.

Example 17:6-(((cis)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The reductive amination was carried out in a similar fashion as forExample 1 using Example B (250 mg, 0.55 mmol) and(cis)-4,4-difluoro-3,5-dimethylpiperidine hydrochloride (203 mg, 1.10mmol) as reactants to afford the title compound (148 mg, 46%). ¹H NMR(500 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.02 (s, 1H), 7.96 (s, 1H), 7.89 (dd,J=8.1, 2.1 Hz, 1H), 7.41 (d, J=2.1 Hz, 1H), 7.36 (dd, J=8.0 Hz, 1H),6.82-6.74 (m, 1H), 5.12 (s, 2H), 4.98 (d, J=6.0 Hz, 2H), 4.90 (d, J=6.1Hz, 2H), 3.76 (s, 2H), 3.52 (s, 2H), 2.81 (d, J=11.3 Hz, 2H), 2.22-2.08(m, 2H), 2.01 (d, J=12.8 Hz, 2H), 0.91 (d, J=6.7 Hz, 6H); LCMS:C₃₀H₃₂F₅N₅O₂ requires: 589, found: m/z=590 [M+H]⁺.

Example 18:(S)-1-((2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindolin-5-yl)methyl)piperidine-3-carbonitrile

The general procedure of Example 1 was followed using(S)-piperidine-3-carbonitrile hydrochloride and gave the title compound(20 mg, 30%). ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.01 (s, 1H), 7.95 (s,1H), 7.94-7.88 (m, 2H), 7.37 (t, J=8.0 Hz, 1H), 7.33 (t, J=2.0 Hz, 1H),6.81-6.74 (m, 1H), 5.04 (d, J=6.1 Hz, 2H), 4.99 (d, J=5.7 Hz, 4H), 3.73(q, J=14.1 Hz, 2H), 3.54 (s, 2H), 2.93 (d, J=5.2 Hz, 1H), 2.88 (s, 3H),2.69 (s, 1H), 2.58 (s, 2H), 2.41 (s, 1H), 1.71 (d, J=76.6 Hz, 4H). MS(ESI) calc'd for (C₂₉H₂₉F₃N₆O₂) [M+H]⁺, 551.2; found, 551.4.

Example 19:6-((cis-2,3-dimethylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The general procedure of Example 1 was followed usingcis-2,3-dimethylpiperidine hydrochloride and gave the title compound (20mg, 30%). ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.02 (s, 1H), 7.96 (s,1H), 7.91 (d, J=6.6 Hz, 2H), 7.37 (t, J=8.0 Hz, 1H), 7.33 (t, J=2.0 Hz,1H), 6.78 (dd, J=7.7, 1.4 Hz, 1H), 5.04 (d, J=6.1 Hz, 2H), 5.01-4.91 (m,4H), 3.78 (m, 2H), 3.54 (s, 2H), 2.88 (s, 3H), 2.84 (m, 1H), 2.55 (s,1H), 2.31 (s, 1H), 1.71-1.25 (m, 2H), 0.97 (d, J=6.7 Hz, 3H), 0.89 (d,J=7.0 Hz, 3H). MS (ESI) calc'd for (C₃₀H₃₄F₃N₅O₂) [M+H]⁺, 554.3; found,554.4.

Example 20 and Example 21:trans-6-{[3,5-bis(trifluoromethyl)piperidin-1-yl]methyl}-2-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)-4-(trifluoromethyl)-3H-isoindol-1-one(20) andcis-6-{[3,5-bis(trifluoromethyl)piperidin-1-yl]methyl}-2-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)-4-(trifluoromethyl)-3H-isoindol-1-one(21)

The reductive amination was carried out in a similar fashion as forExample 1, using Example B (100 mg, 0.22 mmol) and3,5-bis(trifluoromethyl)piperidine (145 mg, 0.66 mmol) as reactants toafford the title compounds. Example 20 (20, 18 mg, 11%): ¹H NMR (500MHz, Acetonitrile-d₃) δ 8.10 (s, 1H), 8.04 (s, 1H), 8.00-7.87 (m, 2H),7.40 (t, J=8.0 Hz, 1H), 7.36 (d, J=2.0 Hz, 1H), 6.84-6.79 (m, 1H),5.05-4.99 (m, 6H), 3.85 (s, 2H), 3.62 (s, 2H), 3.11 (dd, J=11.1, 3.6 Hz,2H), 2.93 (s, 3H), 2.77-2.55 (m, 2H), 2.25-2.16 (m, 2H), 1.99 (s, 1H),1.51-1.40 (m, 1H); LCMS: C₃₀H₂₈F₉N₅O₂ requires: 661, found: m/z=662[M+H]⁺. Example 21 (21, 27 mg, 17%): ¹H NMR (500 MHz, Acetonitrile-d₃) δ8.10 (s, 1H), 8.04 (s, 1H), 8.00-7.87 (m, 2H), 7.40 (t, J=8.0 Hz, 1H),7.36 (d, J=2.0 Hz, 1H), 6.84-6.79 (m, 1H), 5.05-4.99 (m, 6H), 3.85 (s,2H), 3.62 (s, 2H), 3.11 (dd, J=11.1, 3.6 Hz, 2H), 2.93 (s, 3H),2.77-2.55 (m, 2H), 2.25-2.16 (m, 2H), 1.99 (s, 1H), 1.51-1.40 (m, 1H);LCMS: C₃₀H₂₈F₉N₅O₂ requires: 661, found: m/z=662 [M+H]⁺.

Example 22 and Example 23:trans-6-{[3-(hydroxymethyl)-5-methylpiperidin-1-yl]methyl}-2-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)-4-(trifluoromethyl)-3H-isoindol-1-one(22) andcis-6-{[3-(hydroxymethyl)-5-methylpiperidin-1-yl]methyl}-2-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)-4-(trifluoromethyl)-3H-isoindol-1-one(23)

The reductive amination was carried out in a similar fashion as forExample 1, using Example B (100 mg, 0.22 mmol) and(5-methylpiperidin-3-yl)methanol (82 mg, 0.66 mmol) as reactants toafford the title compounds. Example 22 (22, 24 mg, 19%). ¹H NMR (500MHz, Acetonitrile-d₃) δ 8.00 (s, 1H), 7.96-7.85 (m, 3H), 7.38 (dd, J=8.0Hz, 1H), 7.34 (dd, J=2.0 Hz, 1H), 6.78 (d, J=7.8, 1.3 Hz, 1H), 5.05 (d,J=6.1 Hz, 2H), 5.02-4.89 (m, 4H), 3.69-3.59 (m, 2H), 3.57 (d, J=6.6 Hz,2H), 3.55 (s, 2H), 2.88 (s, 3H), 2.63-2.53 (m, 1H), 2.51-2.36 (m, 2H),1.94-1.81 (m, 3H), 1.63-1.51 (m, 2H), 1.29-1.20 (m, 1H), 0.94 (d, J=6.6Hz, 3H); LCMS: C₃₀H₃₄F₃N₅O₃ requires: 569, found: m/z=570 [M+H]⁺.Example 23 (23, 13 mg, 13%): ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.00(s, 1H), 7.95-7.90 (m, 3H), 7.38 (t, J=7.9 Hz, 1H), 7.34 (s, 1H), 6.79(d, J=7.7 Hz, 1H), 5.05 (d, J=6.0 Hz, 2H), 5.00 (d, J=6.5 Hz, 4H),3.73-3.64 (m, 2H), 3.55 (s, 2H), 3.38 (s, 1H), 3.27 (s, 1H), 2.98 (d,J=10.7 Hz, 1H), 2.86 (s, 3H), 2.84 (d, J=10.9 Hz, 1H), 2.53 (s, 2H),1.82-1.69 (m, 3H), 1.62 (s, 2H), 0.88 (d, J=6.3 Hz, 3H); LCMS:C₃₀H₃₄F₃N₅O₃ requires: 569, found: m/z=570 [M+H]⁺.

Example 24:6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(1-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

Step 1: Synthesis of Methyl2-(bromomethyl)-5-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-3-(trifluoromethyl)benzoate:To a stirring solution of 4,4-difluoro-3-methylpiperidinehydrochloride(3.2 g, 18.5 mmol) in DCM (100 mL) was added triethylamine (2.6 mL, 18.4mmol). The solution was cooled to 0° C. then methyl2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (Example B, step 4)(5.0 g, 15.4 mmol) in 10 mL of DCM and sodium triacetoxyborohydride (9.8g, 46.1 mmol) were added. The mixture was allowed to stir at rt forabout 6 h. The reaction was quenched with saturated ammonium chlorideand extracted with DCM. The organic layer was dried, filtered, andconcentrated. The crude material was purified by chromatography A togive the title compound (3.2 g, 47%).

Step 2: Synthesis of5-((1-(3-bromophenyl)cyclopropyl)methyl)-4-methyl-4H-1,2,4-triazole-3-thiol.A mixture of [1-(3-bromophenyl)cyclopropyl]acetic acid (Example 25 Step3) (1.7 g, 6.69 mmol), 1-amino-3-methylthiourea (914 mg, 8.71 mmol), EDC(1.9 g, 10.0 mmol), 1-hydroxy benzotriazole (1.3 g, 10.0 mmol) andtriethylamine (2.0 g, 20.0 mmol) in DMF (10 mL) was stirred at roomtemperature for 2 h. Sodium hydroxide (1 M, 40 mL) solution was added tothe above mixture and stirred at room temperature for another 16 h. ThepH value of the reaction mixture was acidified to ˜5 by the addition ofHCl (1 N). The precipitated solid was collected by filtration, washedwith water, and oven-dried to afford the title compound (820 mg, crude),which was used in the next step without further purification. MS (ESI)calculated for (C₁₃H₁₄BrN₃S) [M+H]⁺, 324; found, 324.

Step 3: Synthesis of3-((1-(3-bromophenyl)cyclopropyl)methyl)-4-methyl-4H-1,2,4-triazole. Toa mixture of5-((1-(3-bromophenyl)cyclopropyl)methyl)-4-methyl-4H-1,2,4-triazole-3-thiol(820 mg, 2.53 mmol) and NaNO₂ (1.7 g, 24.6 mmol) were added HNO₃ (1 N,25 mL) dropwise at 0° C. The mixture was stirred at room temperature for2 h. The reaction was quenched by the addition of saturated sodiumcarbonate aqueous solution, then General Work-up Procedure 1. Theresidue was purified by Chromatography A to afford the title compound(700 mg, 90%). (C₁₃H₁₄BrN₃) [M+H]⁺, 292; found, 292.

Step 4: Synthesis of tert-butyl(3-(1-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)carbamate. A degassed mixture of3-((1-(3-bromophenyl)cyclopropyl)methyl)-4-methyl-4H-1,2,4-triazole (700mg, 2.40 mmol), tert-butyl carbamate (308.7 mg, 2.64 mmol), XPhos Pd G3(405 mg, 0.48 mmol), X-Phos (228 mg, 0.48 mmol), and Cs₂CO₃ (1.6 g, 4.8mmol) in 1,4-dioxane (20 mL) was stirred at 90° C. for 16 h under N₂atmosphere. The reaction was quenched by the addition of water andextracted with EtOAc. The combined organic layers were washed withbrine, dried, and concentrated under vacuum. The crude residue waspurified by (Chromatography B to afford the title compound (500 mg, 57%yield). MS (ESI) calculated for (C₁₈H₂₄N₄O₂) [M+H]⁺, 328; found, 328.

Step 5: Synthesis of6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(1-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The isoindolinone formation reaction was carried out in a manner similarto Example B, step 5 using3-{1-[(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl}aniline (100 mg,0.44 mmol) and methyl2-(bromomethyl)-5-[(4,4-difluoro-3-methylpiperidin-1-yl)methyl]-3-(trifluoromethyl)benzoate(214 mg, 0.48 mmol) as reactants to afford the title compound (75 mg,30%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.25 (s, 1H), 8.02 (s, 1H), 7.96 (s,1H), 7.78 (ddd, J=8.2, 2.4, 1.0 Hz, 1H), 7.75 (t, J=2.0 Hz, 1H), 7.33(dd, J=7.9 Hz, 1H), 7.05 (d, J=7.6, 1.3 Hz, 1H), 5.23-5.08 (m, 2H),3.84-3.66 (m, 2H), 3.34 (s, 3H), 3.12 (s, 2H), 2.76 (dd, J=22.3, 10.2Hz, 2H), 2.33 (t, J=11.2 Hz, 1H), 2.16-1.97 (m, 4H), 0.98-0.89 (m, 7H);LCMS: C₂₉H₃₀F₅N₅O requires: 559, found: m/z=560 [M+H]⁺.

Example 25:6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(1-((S)-fluoro(4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

Step 1: Synthesis of 1-bromo-3-[1-(bromomethyl)cyclopropyl]benzene. To asolution of [1-(3-bromophenyl)cyclopropyl]methanol (WO 2014/201073)(10.0 g, 44.0 mmol) in DCM (100 mL) was added carbon tetrabromide (22.0g, 66.3 mmol) and triphenylphosphine (17.4 g, 66.3 mmol). The mixturewas stirred at rt for 16 h. The mixture was concentrated. The residuewas purified by Chromatography A to afford the title compound (6.5 g,51%).

Step 2: Synthesis of 2-[1-(3-bromophenyl)cyclopropyl]acetonitrile. To asolution of 1-bromo-3-[1-(bromomethyl)cyclopropyl]benzene (6.0 g, 20.6mmol) in DMSO (60 mL) was added sodium cyanide (3.1 g, 62.0 mmol). Themixture was stirred at 60° C. for 3 h. The mixture was diluted withwater, followed by General Work-up Procedure 1 to afford the titlecompound (5.0 g, crude), which was used without purification.

Step 3: Synthesis of 2-[1-(3-bromophenyl)cyclopropyl]acetic acid. To asolution of 2-[1-(3-bromophenyl)cyclopropyl]acetonitrile (5.0 g, 21.1mmol) in ethanol (50 mL) and water (10 mL) was added potassium hydroxide(11.9 g, 212.0 mmol). The mixture was stirred at 80° C. for 16 h. Themixture was acidified with hydrochloric acid to pH=6, followed byGeneral Work-up Procedure 1 to afford the title compound (5.2 g, 96%).

Step 4: Synthesis of ethyl 2-[1-(3-bromophenyl)cyclopropyl]acetate. To asolution of [1-(3-bromophenyl)cyclopropyl]acetic acid (5.2 g, 20.4 mmol)in ethanol (50 mL) was added concentrated HCl (1 mL). The mixture wasstirred at reflux for 2 h. The solvent was evaporated under vacuum. Theresidue was diluted with EtOAc and washed with saturated sodiumbicarbonate aqueous solution. The organic layer was dried andconcentrated under vacuum. The residue was purified by Chromatography Ato afford the title compound (4.0 g, 65%).

Step 5: Synthesis of ethyl2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxyacetate. To a stirringsolution of ethyl 2-[1-(3-bromophenyl)cyclopropyl]acetate (4.0 g, 14.1mmol) in THE (30 mL) was added KHMDS (21.3 mL, 21.30 mmol, 1.0 M in THF)dropwise at −78° C. under N₂ atmosphere. The mixture was stirred for 30min at −78° C. A solution of 3-phenyl-2-(phenylsulfonyl)-1,2-oxaziridine(5.6 g, 21.3 mmol) in THE (10 mL) was added dropwise at −70° C. andstirred for 3 h. The mixture was quenched with saturated NH₄Cl andextracted with EtOAc. The combined organic layers were washed withbrine, dried, and concentrated under vacuum. The residue was purified byChromatography A to afford the title compound (2.5 g, 59%).

Step 6: Synthesis of2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxyacetohydrazide. To a solutionof ethyl 2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxyacetate (2.5 g, 8.3mmol) in ethanol (40 mL) was added N₂H₄.H₂O (10 mL, 80%). The mixturewas stirred at 80° C. for 16 h. The mixture was concentrated undervacuum and used in the next step without purification.

Step 7: Synthesis of2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxy-N-[(methylcarbamothioyl)amino] acetamide. A solution of2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxy acetohydrazide (2.5 g, crude)and methyl isothiocyanate (1.0 g, 13.7 mmol) in THE (30 mL) was stirredat rt for 16 h. The mixture was concentrated under vacuum to afford thetitle compound which was used in the next step without purification.

Step 8: Synthesis of[1-(3-bromophenyl)cyclopropyl](4-methyl-5-sulfanyl-1,2,4-triazol-3-yl)methanol.A suspension of2-[1-(3-bromophenyl)cyclopropyl]-2-hydroxy-N-[(methylcarbamothioyl)amino]-acetamide (2.9 g, crude) in NaOH (aq. 60 mL, 1 M) wasstirred at rt for 16 h. The mixture was acidified to pH ˜3 by HCl (1 N)and extracted with EtOAc. The combined organic layers were washed withbrine, dried, and concentrated under vacuum. The residue was purified byflash column chromatography with EtOAc in petroleum ether to afford thetitle compound (2.4 g, 85% over three steps).

Step 9: Synthesis of[1-(3-bromophenyl)cyclopropyl](4-methyl-1,2,4-triazol-3-yl)methanol. Toa mixture of2-bromo-4-[1-[hydroxy(4-methyl-5-sulfanyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]benzen-1-ylium(2.5 g, 7.2 mmol) and NaNO₂ (4.9 g, 71.6 mmol) was added HNO₃ (1 N, 72.0mL) at 0° C. The mixture was stirred at rt for 3 h. The reaction mixturewas quenched by the addition of NaHCO₃. The precipitated solids werecollected by filtration, washed with water, and dried under vacuum toafford the title compound which was used in the next step withoutpurification.

Step 10: Synthesis of3-[[1-(3-bromophenyl)cyclopropyl](fluoro)methyl]-4-methyl-1,2,4-triazole.To a stirring solution of[1-(3-bromophenyl)cyclopropyl](4-methyl-1,2,4-triazol-3-yl)methanol (2.2g, crude) in DCM (100 mL) was added (N,N-diethylamino)sulfurtrifluoride(4.6 g, 28.6 mmol) dropwise at −78° C. under N₂ atmosphere. The mixturewas stirred at rt for 3 h. The reaction was quenched by the addition ofsaturated aqueous NaHCO₃ solution and extracted with DCM. The combinedorganic layers were washed with brine, dried, and concentrated undervacuum. The residue was purified by Chromatography C to afford the titlecompound (1.5 g, 65%).

Step 11: Synthesis of tert-butylN-(3-[1-[fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclo propyl]phenyl)carbamate. A degassed mixture of3-[[1-(3-bromophenyl)cyclopropyl](fluoro)methyl]-4-methyl-1,2,4-triazole(1.5 g, 4.8 mmol), tert-butyl carbamate (602 mg, 5.1 mmol), XPhos Pd G3(791 mg, 0.94 mmol), XPhos (445 mg, 0.94 mmol), and Cs₂CO₃ (3.0 g, 9.35mmol) in dioxane (40 mL) was stirred at 90° C. for 16 h under N₂atmosphere. When the reaction was completed, the precipitated solidswere filtered off and the filtrate was concentrated under vacuum. Theresidue was diluted with EtOAc and water, and the aqueous layer wasextracted with EtOAc. The combined organic layers were washed withbrine, dried, and concentrated under vacuum. The residue was purified byChromatography A to afford the title compound (1.0 g, 61%).

Step 12: Synthesis of3-[1-[(R)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]anilineand3-[1-[(S)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]aniline.A solution of tert-butylN-(3-[1-[fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]phenyl)carbamate (1.0 g, 2.89 mmol) in HCl (4 M in dioxane, 20 mL) was stirredat rt for 2 h. The solvent was removed under vacuum. The residue waspurified by Chromatography C to afford the title compound (650 mg). Theenantiomers were separated using a chiralpak AS-H column with CO₂ andmethanol as mobile phase to afford3-[1-[(R)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]aniline(242 mg, 34%) and3-[1-[(S)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]aniline(233 mg, 32%).

Step 13:6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(1-((S)-fluoro(4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The isoindolinone formation reaction was carried out in a manner similarto Example B, step 5 using3-[1-[(S)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]aniline(100 mg, 0.41 mmol) and methyl2-(bromomethyl)-5-[(4,4-difluoro-3-methylpiperidin-1-yl)methyl]-3-(trifluoromethyl)benzoate(198 mg, 0.45 mmol) as reactants to afford the title compound (58 mg,25%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.37 (s, 1H), 8.02 (s, 1H), 7.96 (s,1H), 7.85 (ddd, J=8.2, 2.3, 1.0 Hz, 1H), 7.77 (dd, J=1.9 Hz, 1H), 7.36(dd, J=7.9 Hz, 1H), 7.11 (d, J=7.7, 1.2 Hz, 1H), 5.80 (d, J=44.5 Hz,1H), 5.21-5.04 (m, 2H), 3.81-3.72 (m, 2H), 3.24 (s, 3H), 2.84-2.67 (m,2H), 2.33 (t, J=11.1 Hz, 1H), 2.19-1.92 (m, 4H), 1.28-1.22 (m, 2H),1.12-1.02 (m, 2H), 0.94 (d, J=6.5 Hz, 3H); LCMS: C₂₉H₂₉F₆N₅O requires:577, found: m/z=578 [M+H]⁺.

Example 26:6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(1-((R)-fluoro(4-methyl-4H-1,2,4-triazol-3-yl)methyl)cyclopropyl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The isoindolinone formation reaction was carried out in a manner similarto Example B, step 5 using3-[1-[(S)-fluoro(4-methyl-1,2,4-triazol-3-yl)methyl]cyclopropyl]aniline(100 mg, 0.41 mmol) and methyl2-(bromomethyl)-5-[(4,4-difluoro-3-methylpiperidin-1-yl)methyl]-3-(trifluoromethyl)benzoate(198 mg, 0.45 mmol) as reactants to afford the title compound (108 mg,46%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.37 (s, 1H), 8.02 (s, 1H), 7.96 (s,1H), 7.88-7.83 (m, 1H), 7.77 (dd, J=1.9 Hz, 1H), 7.36 (dd, J=7.9 Hz,1H), 7.11 (d, J=7.6, 1.2 Hz, 1H), 5.80 (d, J=44.6 Hz, 1H), 5.21-5.04 (m,2H), 3.81-3.71 (m, 2H), 3.24 (s, 3H), 2.76 (dd, J=25.8, 10.9 Hz, 2H),2.39-2.28 (m, 1H), 2.17-1.93 (m, 4H), 1.30-1.19 (m, 2H), 1.12-1.01 (m,2H), 0.94 (d, J=6.5 Hz, 3H); LCMS: C₂₉H₂₉F₆N₅O requires: 577, found:m/z=578 [M+H]⁺.

Example 27:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(6-(ethylamino)-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-4-(trifluoromethyl)isoindolin-1-one

To a solution of Example L (113 mg, 0.37 mmol),[5-(diphenylphosphanyl)-9,9-dimethyl-9H-xanthen-4-yl]diphenylphosphane(42 mg, 0.07 mmol) and Example D (141 mg, 0.40 mmol) in 1,4-dioxane (4mL) were added Pd(OAc)₂ (8 mg, 0.04 mmol) and cesium carbonate (358 mg,1.10 mmol). The mixture was stirred at 120° C. for 1 h under nitrogen.The solution was filtered through celite and concentrated. The residuewas purified by Chromatography B, followed by Chromatography C to affordthe title compound (42 mg, 19%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.26 (s,1H), 7.99 (s, 1H), 7.95 (s, 1H), 7.42 (d, J=1.2 Hz, 1H), 6.62 (dd, J=5.5Hz, 1H), 5.90 (d, J=1.3 Hz, 1H), 5.16 (s, 2H), 4.91 (d, J=6.1 Hz, 2H),4.79 (d, J=6.0 Hz, 2H), 3.81-3.71 (m, 2H), 3.50 (s, 2H), 3.23 (s, 3H),2.75 (dd, J=21.6, 10.6 Hz, 2H), 2.33 (t, J=11.2 Hz, 2H), 2.17-1.93 (m,5H), 1.14 (t, J=7.1 Hz, 3H), 0.94 (d, J=6.5 Hz, 3H); LCMS: C₃₀H₃₄F₅N₇O₂requires: 619, found: m/z=620 [M+H]⁺.

Example 28:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(6-ethoxy-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-4-(trifluoromethyl)isoindolin-1-one

The coupling reaction was carried out in a similar fashion to Example27, using Example K (70 mg, 0.23 mmol) and Example D (87 mg, 0.23 mmol)as reactants to afford the title compound (43 mg, 31%). ¹H NMR (500 MHz,DMSO-d₆) δ 8.26 (s, 1H), 8.03 (s, 1H), 7.98 (s, 1H), 7.94 (d, J=1.1 Hz,1H), 6.44 (d, J=1.2 Hz, 1H), 5.22 (s, 2H), 4.92 (d, J=6.2 Hz, 2H), 4.84(d, J=6.2 Hz, 2H), 4.37-4.29 (m, 2H), 3.81-3.73 (m, 2H), 3.57 (s, 2H),3.31 (s, 3H), 2.82-2.69 (m, 2H), 2.34 (t, J=11.0 Hz, 1H), 2.15-1.95 (m,4H), 1.36 (t, J=7.0 Hz, 3H), 0.94 (d, J=6.5 Hz, 3H); LCMS: C₃₀H₃₃F₅N₆O₃requires: 620, found: m/z=621 [M+H]⁺.

Example 29:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(oxetan-3-ylamino)pyridin-2-yl)-4-(trifluoromethyl)isoindolin-1-one

Step 1: Synthesis of6-chloro-4-[3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]-N-(oxetan-3-yl)pyridin-2-amine.A degassed mixture Example C (300 mg, 1.0 mmol), oxetan-3-amine (146 mg,2.0 mmol), XantPhos (63 mg, 0.11 mmol), Pd(AcO)₂ (22.5 mg, 0.10 mmol)and K₃PO₄ (638 mg, 3.0 mmol) in dioxane (20 mL) was stirred at 100° C.for 16 h under N₂ atmosphere. The solids were filtered out and thefiltrate was concentrated under vacuum. The residue was dissolved withEtOAc and washed with brine, dried, and concentrated under vacuum. Theresidue was purified by HPLC with acetonitrile in water with 0.1%trifluoroacetic acid to afford the title compound (37 mg, 11%).

Step 2: Synthesis of(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(oxetan-3-ylamino)pyridin-2-yl)-4-(trifluoromethyl)isoindolin-1-one.The coupling reaction was carried out in a similar fashion to Example 27using6-chloro-4-[3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]-N-(oxetan-3-yl)pyridin-2-amine(37 mg, 0.11 mmol) and Example D (38 mg, 0.11 mmol) as reactants toafford the title compound (27 mg, 38%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.27(s, 1H), 7.99 (s, 1H), 7.96 (s, 1H), 7.51 (d, J=1.2 Hz, 1H), 7.38 (d,J=4.0 Hz, 1H), 5.97 (s, 1H), 5.14 (s, 2H), 4.92 (d, J=6.0 Hz, 2H),4.85-4.71 (m, 4H), 4.49 (d, J=4.4 Hz, 2H), 3.83-3.71 (m, 2H), 3.51 (s,2H), 3.24 (s, 3H), 2.84-2.67 (m, 2H), 2.33 (t, J=11.4 Hz, 2H), 2.18-1.92(m, 4H), 0.94 (d, J=6.5 Hz, 3H); LCMS: C₃₁H₃₄F₅N₇O₃ requires: 647,found: m/z=648 [M+H]⁺.

Example 30:(R)-2-(6-cyclopropyl-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

Step 1: Synthesis of2-chloro-6-cyclopropyl-4-[3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]pyridine.A degassed mixture of2,6-dichloro-4-[3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]pyridine(300 mg, 1.0 mmol), cyclopropylboronic acid (344 mg, 4.0 mmol), K₂CO₃(414 mg, 2.9 mmol), and Pd(dppf)Cl₂ (82 mg, 0.11 mmol) in dioxane (20mL) was stirred at 100° C. for 16 h under N₂ atmosphere. The solids werefiltered off and the filtrate was concentrated under vacuum. The residuewas dissolved with EtOAc, washed with brine, dried, and concentratedunder vacuum. The residue was purified by Chromatography C to afford thetitle compound (62 mg, 20%).

Step 2: Synthesis of(R)-2-(6-cyclopropyl-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one.The coupling reaction was carried out in a similar fashion to Example27, using2-chloro-6-cyclopropyl-4-[3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl]pyridine(62 mg, 0.20 mmol) and Example D (71 mg, 0.20 mmol) as reactants toafford the title compound (25 mg, 20%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.26(s, 1H), 8.07 (d, J=1.4 Hz, 1H), 8.02 (s, 1H), 7.97 (s, 1H), 6.94 (d,J=1.5 Hz, 1H), 5.17 (s, 2H), 4.95 (d, J=6.2 Hz, 2H), 4.86 (d, J=6.3 Hz,2H), 3.82-3.72 (m, 2H), 3.58 (s, 2H), 3.30 (s, 3H), 2.82-2.67 (m, 2H),2.33 (t, J=11.2 Hz, 2H), 2.16-1.98 (m, 4H), 1.06-0.79 (m, 7H); LCMS:C₃₁H₃₃F₅N₆O₂ requires: 617, found: m/z=618 [M+H]⁺.

Example 31:(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-(difluoromethyl)-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

The coupling reaction was carried out in a similar fashion to Example27, using Example M (223 mg, 0.65 mmol) and(R)-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one(150 mg, 0.43 mmol) as reactants to afford the title compound (42 mg,16%). ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.39 (s, 1H), 8.02 (s, 1H),7.94 (s, 1H), 7.87 (dd, J=8.0, 2.3 Hz, 1H), 7.59 (dd, J=2.1 Hz, 1H),7.39 (dd, J=7.9 Hz, 1H), 6.99-6.93 (m, 1H), 6.90 (t, J=59.1 Hz, 1H),5.11-4.93 (m, 6H), 3.79-3.66 (m, 4H), 2.93-2.63 (m, 4H), 2.45-2.33 (m,2H), 2.09 (s, 1H), 1.00 (d, J=6.5 Hz, 3H); LCMS: C₂₉H₂₈F₇N₅O₂ requires:611, found: m/z=612 [M+H]⁺.

Example 32: Synthesis of6-cyclopropyl-4-{[(3R)-4,4-difluoro-3-methylpiperidin-1-yl]methyl}-N-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)pyridine-2-carboxamide

1-Propanephosphonic anhydride (0.105 mL, 0.18 mmol) was added to asolution of Example E (45 mg, 0.14 mmol), Example A (35 mg, 0.14 mmol),and 4-methylmorpholine (64 μL, 0.6 mmol) in DMF (0.25 mL) at rt. Thesolution was heated at 45-50° C. for 4 h. The mixture was diluted withwater and acetonitrile, concentrated onto Celite, and purified byChromatography C to afford the title compound (42 mg, 57%): ¹H NMR (500MHz, DMSO-d₆) δ 10.12 (s, 1H), 8.19 (s, 1H), 7.83 (d, J=1.4 Hz, 1H),7.73 (dd, J=8.0, 2.1 Hz, 1H), 7.46-7.38 (m, 2H), 7.27 (t, J=7.9 Hz, 1H),6.66 (dt, J=7.7, 1.3 Hz, 1H), 4.94 (d, J=5.9 Hz, 2H), 4.86 (d, J=6.0 Hz,2H), 3.68-3.56 (m, 2H), 3.49 (s, 2H), 2.92 (s, 3H), 2.79-2.65 (m, 2H),2.33-2.21 (m, 2H), 2.16-1.87 (m, 4H), 1.15 (dt, J=6.1, 3.1 Hz, 2H),1.09-0.99 (m, 2H), 0.94 (d, J=6.7 Hz, 3H); LCMS: C₂₉H₃₄F₂N₆O₂ requires:536, found: m/z=537 [M+H]⁺.

Example 33: Synthesis of6-cyclopropyl-4-{[(3S)-4,4-difluoro-3-methylpiperidin-1-yl]methyl}-N-(3-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}phenyl)pyridine-2-carboxamide

1-Propanephosphonic anhydride (0.105 mL, 0.18 mmol) was added to asolution of Example F (45 mg, 0.14 mmol), Example A (35 mg, 0.14 mmol),and 4-methylmorpholine (64 μL, 0.6 mmol) in N,N-dimethylformamide (0.25mL) at rt. The solution was heated at 45-50° C. for 4 h. The mixture wasdiluted with water and acetonitrile, concentrated onto Celite, andpurified by Chromatography C to afford the title compound (61 mg, 78%):¹H NMR (500 MHz, DMSO-d₆) δ 10.12 (s, 1H), 8.19 (s, 1H), 7.83 (d, J=1.4Hz, 1H), 7.76-7.69 (m, 1H), 7.46-7.39 (m, 2H), 7.27 (t, J=7.9 Hz, 1H),6.66 (dt, J=7.8, 1.4 Hz, 1H), 4.94 (d, J=6.0 Hz, 2H), 4.86 (d, J=6.0 Hz,2H), 3.63 (d, J=3.4 Hz, 2H), 3.49 (s, 2H), 2.92 (s, 3H), 2.72 (dd,J=24.9, 11.5 Hz, 2H), 2.34-2.21 (m, 2H), 2.19-1.87 (m, 4H), 1.15 (dt,J=6.1, 3.1 Hz, 2H), 1.08-1.00 (m, 2H), 0.94 (d, J=6.6 Hz, 3H); LCMS:C₂₉H₃₄F₂N₆O₂ requires: 536, found: m/z=537 [M+H]⁺.

Example 34:(R)-4-Cyclopropyl-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion to Example27, using3-{[3-(3-bromophenyl)oxetan-3-yl]methyl}-4-methyl-1,2,4-triazole (48 mg,0.16 mmol, 1 equiv) and4-cyclopropyl-6-{[(3R)-4,4-difluoro-3-methylpiperidin-1-yl]methyl}-2H,3H-pyrrolo[3,4-c]pyridin-1-one(50 mg, 0.16 mmol, 1 equiv) as reactants. Purification by ChromatographyC then Chromatography B followed by a second Chromatography C affordedthe title compound (7.5 mg, 9%): ¹H NMR (500 MHz, DMSO-d₆) δ 8.21 (s,1H), 7.94 (dd, J=8.2, 2.1 Hz, 1H), 7.51 (s, 1H), 7.45 (t, J=1.9 Hz, 1H),7.37 (t, J=7.9 Hz, 1H), 6.79 (d, J=7.8 Hz, 1H), 5.11 (s, 2H), 4.97 (d,J=6.0 Hz, 2H), 4.91 (d, J=6.1 Hz, 2H), 3.73 (d, J=1.9 Hz, 2H), 3.52 (s,2H), 2.93 (s, 3H), 2.83-2.73 (m, 2H), 2.36 (d, J=8.5 Hz, 1H), 2.25-2.20(m, 1H), 2.10s (d, J=8.2 Hz, 2H), 2.03 (t, J=11.1 Hz, 2H), 1.10 (dd,J=6.6, 3.6 Hz, 4H), 0.94 (d, J=6.2 Hz, 3H); LCMS: C₃₀H₃₄F₂N₆O₂ requires:548, found: m/z=549 [M+H]⁺.

Example 35:(S)-4-Cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion to Example27, using3-{[3-(3-bromophenyl)oxetan-3-yl]methyl}-4-methyl-1,2,4-triazole (50 mg,0.18 mmol, 1 equiv) and4-cyclopropyl-6-{[(3R)-4,4-difluoro-3-methylpiperidin-1-yl]methyl}-2H,3H-pyrrolo[3,4-c]pyridin-1-one(54 mg, 0.18 mmol, 1 equiv) as reactants. Purification by ChromatographyC then Chromatography B followed by a second Chromatography C affordedthe title compound (8.9 mg, 10%): ¹H NMR (500 MHz, DMSO-d₆) δ 8.21 (s,1H), 7.94 (dd, J=8.2, 2.1 Hz, 1H), 7.50-7.42 (m, 2H), 7.37 (t, J=7.9 Hz,1H), 6.78 (d, J=7.9 Hz, 1H), 5.10 (s, 2H), 4.97 (d, J=6.0 Hz, 2H), 4.91(d, J=6.1 Hz, 2H), 3.61 (s, 2H), 3.52 (s, 2H), 2.92 (s, 3H), 2.74 (s,2H), 2.47 (s, 1H), 2.24-2.18 (m, 1H), 1.96 (t, J=10.3 Hz, 1H), 1.64 (dd,J=32.2, 12.2 Hz, 4H), 1.50 (d, J=11.9 Hz, 1H), 1.09 (tq, J=7.9, 5.1, 3.9Hz, 4H), 0.83 (d, J=6.2 Hz, 3H); LCMS: C₃₀H₃₆N₆O₂ requires: 512, found:m/z=513 [M+H]⁺.

Example 36:2-[3-(3-{[4-(difluoromethyl)-1,2,4-triazol-3-yl]methyl}oxetan-3-yl)phenyl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example M (55 mg, 0.16 mmol) and Example N (50 mg, 0.16mmol) as reactants to afford the title compound (14 mg, 15%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 8.39 (s, 1H), 7.99 (s, 1H), 7.92 (s, 1H),7.87 (dd, J=8.4, 2.2 Hz, 1H), 7.58 (dd, J=2.0 Hz, 1H), 7.39 (dd, J=8.0Hz, 1H), 6.95 (dd, J=7.7, 1.5 Hz, 1H), 6.83 (t, J=59.0 Hz, 1H),5.07-5.01 (m, 6H), 3.74 (s, 2H), 3.66 (d, J=13.8 Hz, 2H), 2.79-2.74 (m,2H), 1.80-1.38 (m, 6H), 1.01-0.91 (m, 1H), 0.87 (s, 3H); LCMS:C₂₉H₃₀F₅N₅O₂ requires: 575, found: m/z=576 [M+H]⁺.

Example 37:4-cyclopropyl-2-[3-(3-{[4-(difluoromethyl)-1,2,4-triazol-3-yl]methyl}oxetan-3-yl)phenyl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-3H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example M (60 mg, 0.18 mmol) and Example J (50 mg, 0.18mmol) as reactants to afford the title compound (17 mg, 17%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 8.40 (s, 1H), 7.88 (dd, J=7.9, 2.2 Hz, 1H),7.63 (s, 1H), 7.54 (s, 1H), 7.41 (dd, J=8.0 Hz, 1H), 6.97 (d, J=7.9 Hz,1H), 6.84 (t, J=59.1 Hz, 1H), 5.06-4.97 (m, 6H), 3.74 (s, 2H), 3.62 (s,2H), 2.86-2.71 (m, 3H), 1.83-1.48 (m, 6H), 1.19-1.07 (m, 4H), 0.97-0.89(m, 1H), 0.87 (d, J=6.1 Hz, 3H); LCMS: C₃₀H₃₄F₂N₆O₂ requires: 548,found: m/z=549 [M+H]⁺.

Example 38:4-cyclopropyl-6-{[(3R)-4,4-difluoro-3-methylpiperidin-1-yl]methy}-2-[3-(3-{[4-(difluoromethyl)-1,2,4-triazol-3-yl]methyl}oxetan-3-yl)phenyl]-3H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as Example 27using Example M (54 mg, 0.16 mmol) and Example I (55 mg, 0.16 mmol) asreactants to afford the title compound (22 mg, 24%). ¹H NMR (500 MHz,Acetonitrile-d₃) δ 8.40 (s, 1H), 7.87 (dd, J=8.1, 2.2 Hz, 1H), 7.66-7.62(m, 1H), 7.56 (s, 1H), 7.41 (dd, J=8.0 Hz, 1H), 7.00-6.95 (m, 1H), 6.84(t, J=59.0 Hz, 1H), 5.04 (s, 4H), 4.98 (s, 2H), 3.74 (s, 2H), 3.72 (s,2H), 2.89-2.71 (m, 2H), 2.42 (t, J=11.6 Hz, 1H), 2.14-2.01 (m, 5H),1.20-1.07 (m, 4H), 1.00 (d, J=6.2 Hz, 3H); LCMS: C₃₀H₃₂F₄N₆O₂ requires:584, found: m/z=585 [M+H]⁺.

Example 39:2-(6-ethoxy-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example K (49 mg, 0.16 mmol) and Example N (50 mg, 0.16mmol) as reactants to afford the title compound (16 mg, 17%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 8.01 (s, 1H), 7.96 (s, 1H), 7.95-7.90 (m,2H), 6.31 (s, 1H), 5.21 (s, 2H), 5.00 (d, J=6.2 Hz, 2H), 4.95 (d, J=6.3Hz, 2H), 4.39 (q, J=7.1 Hz, 2H), 3.65 (s, 2H), 3.57 (s, 2H), 3.23 (s,3H), 2.77 (s, 3H), 1.77-1.48 (m, 5H), 1.40 (t, J=7.0 Hz, 3H), 0.97-0.91(m, 1H), 0.87 (d, J=6.1 Hz, 3H); LCMS: C₃₀H₃₅F₃N₆O₃ requires: 584,found: m/z=585 [M+H]⁺.

Example 40:4-cyclopropyl-2-(6-ethoxy-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-3H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example K (54 mg, 0.18 mmol) and Example J (50 mg, 0.18mmol) as reactants to afford the title compound (24 mg, 24%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.97 (s, 1H), 7.93 (s, 1H), 7.55 (s, 1H),6.32 (s, 1H), 5.19 (s, 2H), 5.00 (d, J=6.2 Hz, 2H), 4.96 (d, J=6.3 Hz,2H), 4.43 (q, J=7.0 Hz, 2H), 3.62 (s, 2H), 3.57 (s, 2H), 3.24 (s, 3H),2.85-2.73 (m, 2H), 2.28-2.19 (m, 2H), 1.78-1.54 (m, 3H), 1.40 (t, J=7.0Hz, 3H), 1.19-1.06 (m, 6H), 0.97-0.89 (m, 1H), 0.87 (d, J=6.2 Hz, 3H);LCMS: C₃₁H₃₉N₇O₃ requires: 557, found: m/z=558 [M+H]⁺.

Example 41:4-cyclopropyl-6-{[(3R)-4,4-difluoro-3-methylpiperidin-1-yl]methyl}-2-(6-ethoxy-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-3H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example K (48 mg, 0.16 mmol) and Example I (50 mg, 0.16mmol) as reactants to afford the title compound (28 mg, 30%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.97 (s, 1H), 7.93 (s, 1H), 7.57 (s, 1H),6.33 (s, 1H), 5.20 (s, 2H), 5.00 (d, J=6.2 Hz, 2H), 4.95 (d, J=6.3 Hz,2H), 4.43 (q, J=7.1 Hz, 2H), 3.72 (s, 2H), 3.57 (s, 2H), 3.24 (s, 3H),2.90-2.74 (m, 2H), 2.49-2.37 (m, 1H), 2.27-2.16 (m, 2H), 2.12-2.00 (m,3H), 1.40 (t, J=7.0 Hz, 3H), 1.21-1.06 (m, 4H), 1.00 (d, J=6.2 Hz, 3H);LCMS: C₃₁H₃₇F₂N₇O₃ requires: 593, found: m/z=594 [M+H]⁺.

Example 42:(R)-4-cyclopropyl-6-((4,4-difluoro-3-methylpiperidin-1-yl)methyl)-2-(6-(ethylamino)-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example L (48 mg, 0.16 mmol) and Example I (50 mg, 0.16mmol) as reactants to afford the title compound (14 mg, 14%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.96 (s, 1H), 7.55 (s, 1H), 7.51 (s, 1H),5.90 (s, 1H), 5.19 (s, 1H), 5.15 (s, 2H), 4.98 (d, J=6.1 Hz, 2H), 4.92(d, J=6.1 Hz, 2H), 3.71 (s, 2H), 3.53 (s, 2H), 3.41-3.31 (m, 2H), 3.18(s, 3H), 2.88-2.71 (m, 3H), 2.42 (s, 1H), 2.13-2.00 (m, 4H), 1.21 (t,J=7.2 Hz, 3H), 1.18-1.07 (m, 4H), 1.00 (d, J=6.2 Hz, 3H); LCMS:C₃₁H₃₈F₂N₈O₂ requires: 592, found: m/z=593 [M+H]⁺.

Example 43:4-cyclopropyl-2-[6-(ethylamino)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-3H-pyrrolo[3,4-c]pyridin-1-one

The coupling reaction was carried out in a similar fashion as Example 27using Example L (54 mg, 0.18 mmol) and Example J (50 mg, 0.18 mmol) asreactants to afford the title compound (41 mg, 42%). ¹H NMR (500 MHz,Acetonitrile-d₃) δ 7.96 (s, 1H), 7.55-7.50 (m, 2H), 5.89 (d, J=1.3 Hz,1H), 5.19 (t, J=5.6 Hz, 1H), 5.14 (s, 2H), 4.98 (d, J=6.1 Hz, 2H), 4.92(d, J=6.2 Hz, 2H), 3.62 (s, 2H), 3.53 (s, 2H), 3.36 (qd, J=7.2, 5.5 Hz,2H), 3.18 (s, 3H), 2.80 (t, J=9.1 Hz, 2H), 1.81-1.56 (m, 7H), 1.21 (t,J=7.2 Hz, 3H), 1.18-1.02 (m, 4H), 1.01-0.84 (m, 4H); LCMS: C₃₁H₄₀N₆O₂requires: 556, found: m/z=557 [M+H]⁺.

Example 44:2-[6-(ethylamino)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one

The coupling reaction was carried out in a similar fashion as forExample 27 using Example L (49 mg, 0.16 mmol) and Example N (50 mg, 0.16mmol) as reactants to afford the title compound (17 mg, 18%). ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.98 (s, 1H), 7.95 (s, 1H), 7.91 (s, 1H),7.50 (d, J=1.3 Hz, 1H), 5.87 (d, J=1.3 Hz, 1H), 5.18 (d, J=14.6 Hz, 2H),4.98 (d, J=6.1 Hz, 2H), 4.92 (d, J=6.1 Hz, 2H), 3.64 (s, 2H), 3.53 (s,2H), 3.37-3.27 (m, 3H), 3.17 (s, 3H), 2.84-2.74 (m, 2H), 1.79-1.53 (m,7H), 1.20 (t, J=7.2 Hz, 3H), 0.98-0.90 (m, 1H), 0.87 (d, J=6.1 Hz, 3H);LCMS: C₃₀H₃₅F₃N₆O₃ requires: 583, found: m/z=584 [M+H]⁺.

Example 45:6-((cis-3,5-dimethylpiperidin-1-yl)methyl)-2-(3-(1-methyl-4-(4-methyl-4H-1,2,4-triazol-3-yl)-1H-pyrazol-5-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one

Step 1: Synthesis of ethyl1-methyl-5-(3-nitrophenyl)pyrazole-4-carboxylate. A solution of ethyl5-bromo-1-methylpyrazole-4-carboxylate (1.0 g, 4.29 mmol),3-nitrophenylboronic acid (859 mg, 5.15 mmol), sodium carbonate (909 mg,8.58 mmol dissolved in 4.3 mL water), andbis(triphenylphosphine)palladium(II) dichloride (151 mg, 0.21 mmol) inDME (43 mL) was heated to 90° C. overnight. After this time, GeneralWork-up Procedure 1 was followed and the crude residue was purified byChromatography A to afford the title compound (710 mg, 60% yield).

Step 2: Synthesis of 1-methyl-5-(3-nitrophenyl)pyrazole-4-carboxylicacid. Lithium hydroxide hydrate (119 mg, 2.84 mmol) was added to asolution of ethyl 1-methyl-5-(3-nitrophenyl)pyrazole-4-carboxylate (710mg, 2.58 mmol) in THE (10.3 mL), water (10.3 mL), and methanol (2.0 mL).This solution was heated to 50° C. for 5 h. before being concentrated todryness. This residue was dissolved in EtOAc and washed with 0.1 M HCland the aqueous phase extracted 3× with EtOAc. The combined organicphases were dried, concentrated, and used without purification (600 mg,2.43 mmol).

Step 3: Synthesis of4-methyl-5-[1-methyl-5-(3-nitrophenyl)pyrazol-4-yl]-1,2,4-triazole-3-thiol.To a solution of 1-methyl-5-(3-nitrophenyl)pyrazole-4-carboxylic acid(600 mg, 2.43 mmol), 4-methyl-3-thiosemicarbazide (319 mg, 3.03 mmol),and N,N-diisopropylethylamine (0.42 mL, 2.43 mmol) in DMF (6.4 mL) wasadded HATU (1.10 g, 2.91 mmol). The reaction was stirred for 4 h beforethe addition of 1 M sodium hydroxide (5.1 mL). This solution was stirredat 50° C. overnight. After cooling to rt, saturated ammonium chloride(30 mL) was added and the mixture stirred 15 min. The solids werefiltered, rinsed with water, and dried under vacuum to provide the titlecompound (600 mg, 1.90 mmol).

Step 4: Synthesis of3-[2-methyl-4-(4-methyl-1,2,4-triazol-3-yl)pyrazol-3-yl]aniline. Thereduction was carried out in a similar fashion to Example A step 6,using4-methyl-5-[1-methyl-5-(3-nitrophenyl)pyrazol-4-yl]-1,2,4-triazole-3-thiol(200 mg, 0.63 mmol) to afford the title compound.

Step 5: Synthesis of2-{3-[2-methyl-4-(4-methyl-1,2,4-triazol-3-yl)pyrazol-3-yl]phenyl}-3-oxo-7-(trifluoromethyl)-1H-isoindole-5-carbaldehyde.To a cool (0° C.) solution of3-[2-methyl-4-(4-methyl-1,2,4-triazol-3-yl)pyrazol-3-yl]aniline (67 mg,0.26 mmol), methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate(85 mg, 0.26 mmol) in acetonitrile (2.32 mL) and water (0.2 mL) wasadded silver nitrate (58 mg, 0.34 mmol) dissolved in 1 mL water. Thereaction was stirred for 40 h at room temperature at which point solidsodium bicarbonate was added until the solution was pH 8. The mixturewas then filtered through Celite, and rinsed with acetonitrile (30 mL)followed by a DCM-EtOAc mixture (30 mL, 9:1). The organic layer wasseparated, dried, and concentrated. The crude residue was purified byChromatography B to afford the title compound (56 mg, 46%).

Step 6. Synthesis of6-(((3S,5R)-3,5-dimethylpiperidin-1-yl)methyl)-2-(3-(1-methyl-4-(4-methyl-4H-1,2,4-triazol-3-yl)-1H-pyrazol-5-yl)phenyl)-4-(trifluoromethyl)isoindolin-1-one.The reductive amination was carried out as in Example 1, using2-(3-(1-methyl-4-(4-methyl-4H-1,2,4-triazol-3-yl)-1H-pyrazol-5-yl)phenyl)-3-oxo-7-(trifluoromethyl)isoindoline-5-carbaldehyde(35 mg, 0.08 mmol) and cis-3,5-dimethylpiperidine (17 mg, 0.15 mmol).The title compound was obtained after Chromatography C (18 mg, 0.03mmol). LCMS: C₃₀H₃₂F₃N₇O requires 563.2, found 564.5 [M+H]⁺. ¹H NMR (500MHz, Acetonitrile-d₃) δ 8.11 (s, 1H), 8.03-7.95 (m, 3H), 7.88 (s, 1H),7.74 (s, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.20 (d, J=7.7 Hz, 1H), 5.03 (s,2H), 3.90 (s, 3H), 3.62 (s, 2H), 3.36 (s, 3H), 2.81-2.74 (m, 2H),1.75-1.60 (m, 3H), 1.53 (t, J=10.9 Hz, 2H), 0.82 (d, J=6.6 Hz, 6H), 0.54(q, J=11.8 Hz, 1H).

Example 46:(R)-6-cyclopropyl-5-(17-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)-15-oxo-5,8,11-trioxa-2,14-diazaheptadecyl)-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide

Step 1: Synthesis of methyl6-cyclopropyl-5-(hydroxymethyl)pyridine-2-carboxylate. A mixture ofmethyl 6-chloro-5-(hydroxymethyl)pyridine-2-carboxylate (Gangadasu, B.et al., Tetrahedron. 2006, 62, 8398-8403) (1.0 g, 5.0 mmol), potassiumcyclopropyltrifluoroboranuide (2.1 g, 14.1 mmol), Pd(dppf)Cl₂ (770 mg,1.05 mmol), and K₃PO4 (3.8 g, 18.1 mmol) in toluene (40 mL) and water (4mL) was heated to 100° C. for 16 h under nitrogen. The mixture wascooled to rt and then filtered. The filtrate was evaporated undervacuum. The residue was purified by Chromatography A to afford the titlecompound (834.0 mg, 81%). LCMS: C₁₁H₁₃NO₃ requires 207.2, found 207.9[M+H]⁺.

Step 2: Synthesis of6-cyclopropyl-5-(hydroxymethyl)pyridine-2-carboxylic acid. A mixture ofmethyl 6-cyclopropyl-5-(hydroxymethyl)pyridine-2-carboxylate (170.0 mg,0.82 mmol) and LiOH (45.0 mg, 1.88 mmol) in THE (6 mL) and water (2 mL)was stirred at rt for 3 h. The pH of the mixture was adjusted to ˜5 withHCl (1 N). The mixture was evaporated under vacuum to afford the titlecompound (200.0 mg, crude), which was used without purification. MS(ESI) calculated for (C₁₀H₁₁NO₃) [M+H]⁺, 194.1, found, 193.9.

Step 3: Synthesis of6-cyclopropyl-5-(hydroxymethyl)-N-[3-[(2R)-1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl]phenyl]pyridine-2-carboxamide.To a mixture of 6-cyclopropyl-5-(hydroxymethyl)pyridine-2-carboxylicacid (200.0 mg, crude) in DMF (3 mL) were added DIEA (1 mL, 6.05 mmol),3-[(2R)-1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl]aniline (173.6 mg,0.80 mmol), and HATU (883.0 mg, 2.32 mmol). The mixture was stirred atrt for 2 h. The mixture was purified by Chromatography C, then purifiedby Prep-HPLC to afford the title compound (31.6 mg, 10%). MS (ESI)calculated for (C₂₂H₂₅N₅O₂) [M+H]⁺, 392.2, found, 392.2.

Step 4: Synthesis of(R)-6-cyclopropyl-5-formyl-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide.To a solution of(R)-6-cyclopropyl-5-(hydroxymethyl)-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide(3.1 g, 7.9 mmol) in methylene chloride (30 mL) was added Dess-Martinreagent (4.0 g, 9.5 mmol) at 0° C. The mixture was stirred at 0° C. for1 h, and then quenched by the addition of saturated aqueous NaHCO₃. Theaqueous phase was extracted with EtOAc. The organic layers werecombined, washed with brine, dried, and filtered. The filtrate wasconcentrated. The residue was purified by Chromatography B to afford thetitle compound (1.8 g, 58%). MS (ESI) calculated for (C₂₂H₂₃N₅O₂)[M+H]⁺, 390.2; found 390.2.

Step 5. Synthesis of(R)-5-(13-amino-5,8,11-trioxa-2-azatridecyl)-6-cyclopropyl-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide.Sodium triacetoxyborohydride (0.05 g, 0.23 mmol) was added to a DCM(1.00 mL) solution containing tert-butylN-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate (45 mg, 0.15mmol) and(R)-6-cyclopropyl-5-formyl-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide(60 mg, 0.15 mmol). The mixture was stirred at room temperature for 3 h.After concentration, the crude reaction mixture was purified by reversephase preparative HPLC (Waters 5 mM CSH C18 column, 50×50 mm), elutingwith acetonitrile in water with 0.1% TFA. The desired fractions werecombined and concentrated to give tert-butyl(R)-(1-(2-cyclopropyl-6-((3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)carbamoyl)pyridin-3-yl)-5,8,11-trioxa-2-azatridecan-13-yl)carbamate,which was treated with DCM/TFA 1:1 solution at room temperature. After 1h the reaction was concentrated to afford the title compound (51 mg);LCMS: C₃₀H₄₃N₇O₄ requires m/z=565, found 566 [M+H]⁺.

Step 6. Synthesis of(R)-6-cyclopropyl-5-(17-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)-15-oxo-5,8,11-trioxa-2,14-diazaheptadecyl)-N-(3-(1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)phenyl)picolinamide.Triethylamine (0.01 mL, 6.08 mg, 0.06 mmol) was added to a DMF solution(1 mL) containing HATU (17 mg, 0.05 mmol) and3-(5,5-difluoro-7,9-dimethyl-5H-524,624-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanoicacid (9 mg, 0.03 mmol). After stirring for 5 min at room temperature,5-(13-amino-5,8,11-trioxa-2-azatridecan-1-yl)-6-cyclopropyl-N-{3-[(2R)-1-(4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl]phenyl}pyridine-2-carboxamide(17 mg, 0.03 mmol) was added, and the resulting solution was stirred atrt for 4 h. The crude reaction mixture was purified by reverse phasepreparative HPLC, eluting with acetonitrile in water with 0.1% TFA, toafford the title compound. ¹H NMR (500 MHz, Methanol-d₄) δ 8.00 (s, 2H),7.67 (t, J=2.0 Hz, 1H), 7.55-7.45 (m, 1H), 7.38 (s, 1H), 7.33 (t, J=7.9Hz, 1H), 7.05 (d, J=7.7 Hz, 1H), 6.97 (d, J=4.0 Hz, 1H), 4.57 (s, 2H),3.82 (dd, J=5.7, 4.2 Hz, 2H), 3.68 (hd, J=3.9, 2.6 Hz, 4H), 3.65-3.62(m, 3H), 3.61 (s, 3H), 3.60-3.56 (m, 2H), 3.48 (t, J=5.6 Hz, 3H), 3.39(q, J=6.2, 5.6 Hz, 3H), 3.34 (s, 4H), 3.18 (t, J=7.8 Hz, 3H), 2.59 (t,J=7.7 Hz, 2H), 2.47 (s, 3H), 2.35 (tt, J=8.3, 4.7 Hz, 1H), 2.24 (s, 3H),1.46 (d, J=6.7 Hz, 3H), 1.37-1.28 (m, 2H), 1.19 (dt, J=8.2, 3.3 Hz, 3H);LCMS: C₄₄H₅₆BF₂N₉O₅ requires m/z=840, found 841 [M+H]⁺.

Example 47:2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one

To a solution of Example C (210 mg, 0.70 mmol),[5-(diphenylphosphanyl)-9,9-dimethyl-9H-xanthen-4-yl]diphenylphosphane(81 mg, 0.14 mmol), and Example N (219 mg, 0.70 mmol) in 1,4-dioxane (7mL) were added (acetyloxy)palladio acetate (15 mg, 0.07 mmol) and cesiumcarbonate (0.69 g, 2.11 mmol). The mixture was stirred at 90° C. for 1 hunder nitrogen. The residue was filtered through celite andconcentrated. The residue was purified by Chromatography B to afford thetitle compound (152 mg, 38%). ¹H NMR (500 MHz, Acetonitrile-d₃) δ 8.41(s, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.33 (d, J=4.0 Hz, 1H), 7.11 (s,1H), 5.17 (s, 2H), 5.05-4.95 (m, 4H), 3.81-3.66 (m, 3H), 3.61 (s, 2H),3.34 (s, 3H), 2.95-2.75 (m, 2H), 1.85-1.55 (m, 4H), 1.05-0.92 (m, 2H),0.89 (s, 3H); LCMS: C₂₈H₃₀ClF₃N₆O₂ requires: 574, found: m/z=575 [M+H]⁺.

Example 48:(S)-2-(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one

To a solution of Example 47 (150 mg, 0.26 mmol) in MeOH (5 mL) under anatmosphere of nitrogen was added palladium on carbon (10 wt %, 25 mg).The suspension was then put under an atmosphere of hydrogen and stirredfor 12 h. The suspension was filtered through celite and thenconcentrated. The residue was purified by prep-HPLC using acetonitrilein water to afford the title compound (52 mg, 33%). ¹H NMR (500 MHz,Acetonitrile-d₃) δ 8.51-8.45 (m, 3H), 8.39 (d, J=5.2 Hz, 1H), 8.35 (d,J=1.6 Hz, 1H), 7.02 (dd, J=5.2, 1.6 Hz, 1H), 5.25 (s, 2H), 5.08 (d,J=6.4 Hz, 2H), 5.00 (d, J=6.5 Hz, 2H), 4.37-4.28 (m, 2H), 3.77 (s, 2H),3.36 (s, 1H), 3.23 (d, J=12.0 Hz, 2H), 2.74 (q, J=11.1, 10.6 Hz, 1H),2.45 (q, J=11.3 Hz, 1H), 2.31-2.20 (m, 1H), 2.13 (q, J=13.4 Hz, 1H),1.84 (d, J=13.3 Hz, 1H), 1.12 (qd, J=13.6, 13.1, 4.0 Hz, 1H), 0.92 (d,J=6.7 Hz, 3H); LCMS: C₂₈H₃₁F₃N₆O₂ requires: 540, found: m/z=541 [M+H]⁺.

Example 49 and Example 50:4-cyclopropyl-6-((S)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(49) and4-cyclopropyl-6-((R)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(50)

Step 1: Synthesis of4-cyclopropyl-6-(1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.A mixture of Example O (102 mg, 0.231 mmol),(R)-4,4-difluoro-3-methylpiperidine hydrochloride (61.7 mg, 0.360 mmol),and triethylamine (50 μL, 0.36 mmol) in methanol was microwaved at 100°C. for 1 minute. Sodium cyanoborohydride (22.1 mg, 0.352 mmol) was addedand further microwaved at 80° C. for 30 minutes and at 200° C. for 2hours. More sodium cyanoborohydride (31.0 mg, 0.181 mmol) andtriethylamine (25 μL, 0.18 mmol) was added and microwaved at 100° C. for2 hours. Another portion of sodium cyanoborohydride (27.7 mg, 0.161mmol) and triethylamine (22.5 μL, 0.161 mmol) were added and microwavedat 100° C. for 30 minutes. Saturated sodium bicarbonate solution, water,and dichloromethane were added to the reaction mixture. The desiredproduct was extracted with methanol in dichloromethane (4:1) four times.The organic layers were dried, filtered, and concentrated. Uponpurification with reverse phase HPLC,4-cyclopropyl-6-(1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-onewas obtained (21.7 mg, 17% yield).

Step 2: Separation of4-cyclopropyl-6-((S)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-oneand4-cyclopropyl-6-((R)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.(±)-4-cyclopropyl-6-(1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-onewas SFC separated using a chiralpak AZ column and a mixture of isopropylalcohol: acetonitrile (1:1) with 0.1% diethylamine and CO₂ to give4-cyclopropyl-6-((S)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(5.6 mg) with shorter retention time and4-cyclopropyl-6-((R)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(6.2 mg) with longer retention time.

4-cyclopropyl-6-((S)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one:MS (ESI) calculated for (C₃₁H₃₆F₂N₆O₂) [M+1]⁺, 564; found, 564. ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.90 (d, J=7.2 Hz, 2H), 7.45 (s, 1H),7.40-7.31 (m, 2H), 6.82-6.76 (m, 1H), 5.02 (d, J=6.0 Hz, 2H), 4.97 (d,J=6.0 Hz, 2H), 4.91 (s, 2H), 3.84 (q, J=6.9 Hz, 1H), 3.52 (s, 2H), 2.87(s, 3H), 2.80 (d, J=15.8 Hz, 2H), 2.32 (t, J=11.4 Hz, 1H), 2.19-1.79 (m,5H), 1.38 (d, J=6.9 Hz, 3H), 1.21-1.11 (m, 2H), 1.09 (dd, J=8.0, 3.3 Hz,2H), 0.95 (d, J=6.2 Hz, 3H).

4-cyclopropyl-6-((R)-1-((R)-4,4-difluoro-3-methylpiperidin-1-yl)ethyl)-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one:MS (ESI) calculated for (C₃₁H₃₆F₂N₆O₂) [M+1]⁺, 564; found, 564. ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.90 (d, J=9.4 Hz, 2H), 7.46 (s, 1H),7.40-7.32 (m, 2H), 6.79 (d, J=7.7 Hz, 1H), 5.02 (d, J=5.9 Hz, 2H), 4.97(d, J=6.1 Hz, 2H), 4.91 (s, 2H), 3.84 (q, J=6.9 Hz, 1H), 3.52 (s, 2H),2.87 (s, 3H), 2.87-2.77 (m, 2H), 2.31 (t, J=11.5 Hz, 1H), 2.19-1.79 (m,5H), 1.38 (d, J=6.8 Hz, 3H), 1.15 (dddd, J=17.7, 13.3, 8.6, 4.4 Hz, 2H),1.09 (dd, J=8.0, 3.6 Hz, 2H), 0.93 (d, J=6.0 Hz, 3H).

Example 51 and Example 52:4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((S)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(51) and4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((R)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(52)

Step 1: Synthesis of4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-(1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.Reductive amination was performed in a similar fashion as to step 1 ofExample 49 using (S)-3-methylpiperidine hydrochloride to give4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-(1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(19.8 mg, 0.0376 mmol, 24% yield).

Step 2: Separation of4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((S)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-oneand4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((R)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one.(±)-4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-(1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-onewas SFC separated using a chiralpak AZ column and a mixture of isopropylalcohol:acetonitrile (1:1) with 0.1% diethylamine and CO₂ to give4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((S)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(6.1 mg) with shorter retention time and4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((R)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one(6.5 mg) with longer retention time.

4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((S)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one:MS (ESI) calculated for (C₃₁H₃₈N₆O₂) [M+1]⁺, 528; found, 528. ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.94-7.86 (m, 2H), 7.46 (s, 1H), 7.39-7.31(m, 2H), 6.78 (d, J=7.5 Hz, 1H), 5.02 (d, J=6.0 Hz, 2H), 4.97 (d, J=6.0Hz, 2H), 4.91 (s, 2H), 3.69 (q, J=6.9 Hz, 1H), 3.52 (s, 2H), 2.87 (s,3H), 2.82 (d, J=10.0 Hz, 1H), 2.74 (d, J=11.1 Hz, 1H), 1.71-1.52 (m,4H), 1.53-1.39 (m, 1H), 1.34 (d, J=6.8 Hz, 3H), 1.24-1.10 (m, 2H), 1.08(dd, J=8.2, 3.0 Hz, 2H), 0.83 (d, J=6.3 Hz, 3H), 0.81-0.76 (m, 1H).

4-cyclopropyl-2-(3-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)phenyl)-6-((R)-1-((S)-3-methylpiperidin-1-yl)ethyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one:MS (ESI) calculated for (C₃₁H₃₈N₆O₂) [M+1]⁺, 528; found, 528. ¹H NMR(500 MHz, Acetonitrile-d₃) δ 7.95-7.86 (m, 2H), 7.46 (s, 1H), 7.40-7.32(m, 2H), 6.82-6.75 (m, 1H), 5.02 (d, J=5.9 Hz, 2H), 4.97 (d, J=6.0 Hz,2H), 4.91 (s, 2H), 3.68 (q, J=6.8 Hz, 1H), 3.52 (s, 2H), 2.87 (s, 3H),2.84 (d, J 11.9 Hz, 1H), 2.74 (d, J=9.8 Hz, 1H), 1.72-1.42 (m, 5H), 1.34(d, J=6.8 Hz, 3H), 1.22-1.10 (m, 2H), 1.08 (dd, J=8.1, 3.4 Hz, 2H), 0.79(d, J=6.2 Hz, 4H), 0.83-0.76 (m, 1H).

Biological Examples

The following abbreviations apply: ACT (adoptive cell therapy); AUC(area under curve); Cmpd (compound); CP (cell proliferation); E/T(Effector:Target cell ratio); ID (identification); MFI (meanfluorescence intensity); mpk (milligram per kilogram); PBMC (peripheralblood mononuclear cells); TIL (tumor infiltrating lymphocyte); DPBS(Dulbecco's phosphate-buffered saline); and Ub (ubiquitin).

Biological Example 1: Evaluation of Cbl-b Inhibition by CandidateInhibitors

Candidate compounds were evaluated for their ability to bind and inhibitCbl-b, an E3 ubiquitin-protein ligase, as evidenced by their ability todisplace a fluorophore-labeled probe (Example 46) bound to Cbl-b.

Materials and Methods

Cbl-b Displacement Assay (Cbl-b Inhibition Assay)

The ability of candidate compounds to displace a known inhibitor andthereby inhibit Cbl-b activity was measured by monitoring theinteraction of Cbl-b with a fluorophore-labeled probe in the presence ofthe candidate compound. A truncated variant of Cbl-b (UniProt numberQ13191; SEQ ID NO:1) containing an Avitag at its N-terminus wasco-expressed with BirA biotin ligase and purified using a standardprotocol (see Dou et al., Nature Structural and Molecular Biology 8:982-987, 2013; Avidity LLC).

Cb1-b amino acid residues 36-427: (SEQ ID NO: 1)PKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPII VDPFD

Fluorescently-labeled inhibitor probe was synthesized and tagged withBODIPY FL (Example 46). Cbl-b displacement assays were performed in a384-well plate at room temperature in a 10 μL reaction volume bypre-incubating 0.5 nM Cbl-b or 0.125 nM Cbl-b (final concentration,indicated as “High” and “Low”, respectively) in an assay buffercontaining 20 mM HEPES pH 7.5, 150 mM NaCl, 0.0100 Triton X-100, 0.01% oBSA, and 0.5 mM TCEP in the presence of a candidate compound in 100 DMSO(final concentration) for 1 hour. After incubation in the presence ofthe candidate compound, the plate was incubated for an additional 1 hourin the presence of an approximate EC₄₀ binding saturation consisting of150 nM fluorescently-labeled inhibitor probe and 2 nMStreptavidin-Terbium (Cisbio) (final concentrations). Following the 1hour incubation, the plates were read for TR-FRET signal at 520/620 nMusing an Envision plate reader (Perkin Elmer). The presence of a TR-FRETsignal indicated that the probe was not displaced from Cbl-b by thecompound candidate. The absence of a FRET signal indicated that theprobe was displaced from Cbl-b by the compound candidate.

Compounds were ranked into bins A through C as follows for IC₅₀: Aindicates <1 nM; B indicates 1 nM-5 nM; C indicates 5.1-100 nM.

TABLE 2 Cbl-b inhibition by tested compounds Cbl-b Cbl-b Cmpd activityactivity No. IC₅₀ (High) IC₅₀ (Low) 1 A 2 A 3 B 4 B 5 C 6 C 7 C 8 C 9 C10 A 11 B 12 C 13 B 14 B 15 B 16 B 17 B 18 B 19 B 20 B 21 C 22 A 23 A 24A 25 B 26 B 27 C 28 B 29 C 30 C 31 B 32 A 33 B 34 B 35 B 36 A 37 B 38 B39 A 40 A 41 B 42 B 43 B 44 A 45 B 47 C 48 B 49 A 50 A 51 A 52 B Blankcell indicates data not available.

Biological Example 2: Evaluation of T-Cell Activation by Cbl-bInhibitors

Loss of Cbl-b function in both T-cells and mice by genetic knockout ofthe cbl-b gene results in loss of the CD28 co-stimulation requirementfor T-cell activation and T-cell resistance to anergy (see Bachmaier etal., Nature, 403: 211-216, 2000; and Jeon et al., Immunity, 21: 167-177,2004). Cbl-b inhibitors described herein were evaluated for theirability to activate T-cells.

Materials and Methods

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.The cells were rested overnight at 37° C. 5% CO₂. The Cbl-inhibitor wasadded to 1×10⁵ cells per well and the plate was incubated for one hourat 37° C. in 5% CO₂ at the concentrations indicated (Table 3) with afinal DMSO concentration of <0.1%. For samples stimulated with anti-CD3antibody and anti-CD28 antibody (anti-CD3/anti-CD28), the Cbl-binhibitor concentrations tested were 1 μM, and 0.3 μM. For samplesstimulated with anti-CD3 antibody alone (anti-CD3), the Cbl-b inhibitorconcentrations tested were 3 μM, and 1 μM. Following incubation with theCbl-b inhibitor, primary human total T-cells were stimulated with eitherplate bound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3antibody (OKT3) with soluble anti-CD28 antibody (28.2) (LifeTechnologies). To prepare plates with plate bound anti-CD3 antibody(OKT3), 96-well round bottom tissue culture plates were coated with 100μL of anti-CD3 antibody (OKT3) at 10 pg/mL for 4 hours at 37° C. 5% CO₂in phosphate buffered saline (PBS). The plates were washed with PBS onceprior to adding the cells with or without soluble anti-CD28 antibody(28.2) to each well at a final concentration of 5 μg/mL. Cells werestimulated for 48 hours prior to harvesting the cell free supernatantand staining the cell population for surface marker assessment by flowcytometry. Supernatants were analyzed for cytokine secretion, includingIL-2 by ELISA (R&D Systems, Peprotech or Life Technologies) or Luminexmultiplex kits (Procarta Life Technologies) following the manufacturer'sprotocol. Cells were stained with anti-CD25 antibody (BD Biosciences) toassess levels of surface marker of activation.

Results

Readouts were reported as fold change over baseline. Baseline for thisstudy was the measurement obtained from total human T-cells stimulatedwith anti-CD3 antibody and with soluble anti-CD28 antibody, wherein thecells were not incubated with a Cbl-b inhibitor (Table 3). For T-cellsstimulated with anti-CD3/anti-CD28, changes greater than 2.5-fold overbaseline for IL-2 secretion and greater than 1.3-fold over baseline forCD25 surface staining were considered significant and a positiveresponse (Table 3). For T-cells stimulated with anti-CD3 alone, changesgreater than 0.1-fold over baseline for IL-2 secretion and greater than0.6-fold over baseline for CD25 surface staining were consideredsignificant and a positive response (Table 3). Compounds were rankedinto bins according to their readouts as follows:

For IL-2 secretion with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates ≤10 fold, B indicates 11-15fold, A indicates >15 fold;

For IL-2 secretion with anti-CD3 antibody stimulation the bins are: Cindicates ≤0.33 fold, B indicates 0.34-0.66 fold, A indicates >0.66fold;

For CD25 staining with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates ≤1.24 fold, B indicates1.25-1.39 fold, A indicates >1.39 fold;

For CD25 staining with anti-CD3 antibody stimulation the bins are: Cindicates ≤1.04 fold, B indicates 1.05-1.14 fold, and A indicates >1.14fold.

TABLE 3 T-cell activation as assessed by IL-2 secretion and CD25 surfacestaining as a consequence of stimulation with anti-CD3, or anti-CD3 andanti-CD28 IL-2 secretion IL-2 secretion CD25 staining CD25 staining CmpdCD3/CD28 CD3 CD3/CD28 CD3 No. 1 μM 0.3 μM 3 μM l μM 1 μM 0.3 μM 3 μM lμM 1 A A A A c C c C 2 A A A B c C B B 3 A B B B B B B B 4 A A B B A A AA 5 A B B C A A A A 6 A C B C A A A A 7 B B B C A A A A 8 A A B B B B AA 9 A B B B A B A A 10 B B C C B B B B 11 B C C C B B C C 12 B C C C B BB C 13 B B B B C C C B 14 B C B B C C C B 15 B C B C C C B B 16 C C C CC C B C 17 A B A A B A A A 18 B C B C B B A A 19 B C B C B B B B 21 C CC C B B C C 22 A A A A A A C C 23 A A A A A A B B 27 A A A A C C C C 28A A B B C B C B 29 B B B B C C C C 30 A A B B C C C C 31 A A A A A A C C32 A A B B C C B B 33 B B B C C C B B 35 A B A B B C A B 36 A A A A A AA A 37 A A A A A A A A 38 A A A A A A A A 39 A A A A A A A A 40 A A A AA A A A 41 A A A A A A A A 42 A A A A A A A A 43 A A A A A A A A 44 A AA A A A A A 45 B B B B C B C C 47 A A A A A A A A 48 A A A A A A A A 49A A A A A A A A 50 A A A A A A A A 51 A A A A A A A A 52 A A A A A A A A

Biological Example 3: Evaluation of Immunomodulatory Effects of Cbl-bInhibitors

Cbl-b inhibitors identified from screening assays demonstrated theability to activate total human T-cells in vitro as evidenced byenhanced IL-2 secretion and expression of the CD25 surface activationmarker. Further in vitro studies were conducted to assess additionalcytokine secretion by T-cells and expression of surface activationmarkers on T-cells. Additional immunomodulatory effects on T-cellscontacted with the Cbl-b inhibitors described herein were assessed, suchas the ability of a Cbl-b inhibitor to increase T-cell proliferation,decrease T-cell exhaustion, and decrease T-cell anergy. The ability ofCbl-b inhibitors, such as those described herein, to activate T-cells invivo was also assessed. Other immunomodulatory effects by the Cbl-binhibitors were assessed, such as the ability of Cbl-b inhibitors toactivate B-cells and NK-cells.

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.For measurement of cell proliferation, cells were labeled with CellTrace Violet (Invitrogen) following the manufacturer's protocol prior toactivation by stimulation with anti-CD3 antibody alone or in combinationwith anti-CD28 antibody. The Cbl-b inhibitor was added to 1×10⁵ cellsper well at multiple concentrations (e.g., 10 μM, 1.11 μM, or 0.123 μM)with a final DMSO concentration of <0.1%. The plate was incubated forone hour at 37° C. in 5% CO₂. Following incubation with the Cbl-binhibitor, primary human total T-cells were stimulated with either platebound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3 antibody(OKT3) with soluble anti-CD28 antibody (28.2) (Life Technologies). Toprepare plates with plate bound anti-CD3 antibody (OKT3), 96-well roundbottom tissue culture plates were coated with 100 μL of anti-CD3antibody (OKT3) at 10 μg/mL for 4 hours at 37° C. 5% CO₂ in phosphatebuffered saline (PBS). The plates were washed with PBS prior to addingthe cells with or without soluble anti-CD28 antibody (28.2) to each wellat a final concentration of 5 μg/mL. Cells were stimulated for 48 hoursprior to harvesting the cell free supernatant and staining the cellpopulation for surface marker assessment by flow cytometry. Supernatantswere analyzed for cytokine secretion (e.g., GM-CSF, IFNγ, and TNFα) byELISA (R&D Systems, Peprotech or Life Technologies) or Luminex multiplexkits (Procarta Life Technologies) following the manufacturer's protocol.Cells were stained with anti-CD69 (BD Biosciences) to assess levels ofsurface markers of activation. Proliferation was measured by flowcytometry and data was analyzed with FlowJo v7.6.5 or v10. Readouts werereported as fold change over baseline. In some embodiments, baseline wasthe measurement obtained from total human T-cells stimulated withanti-CD3 antibody alone, wherein the cells were not incubated with aCbl-b inhibitor. In some embodiments, baseline was the measurementobtained from total human T-cells stimulated with anti-CD3 antibody andanti-CD28 antibody, where the cells were not incubated with a Cbl-binhibitor.

Cbl-b inhibitor effects on primary human T-cells were also evaluated inthe context of an allogenic mixed lymphocyte reaction (MLR). Allogenicimmature dendritic cells were generated under the following conditions.Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Monocytes were isolated from the PMBCsutilizing positive selection with a commercial kit following themanufacturer's protocol (Stemcell Technologies Catalog #17858) toyield>95% CD14+ cells as assessed by flow cytometry. Monocytes werecultured with 30 ng/mL of recombinant human GM-CSF and 20 ng/mL ofrecombinant human IL-4 for seven days to generate immature dendriticcells. Monocytes and T-cells were either isolated fresh from peripheralblood or thawed from frozen stocks. Human T-cells were isolated, labeledwith CFSE, and incubated with inhibitors as described above. The Cbl-binhibitor was added to 1×10′ T-cells in coculture with 2×10³ allogenicimmature dendritic cells per well at multiple concentrations (e.g., 10μM, or 1.11 μM) with a final DMSO concentration of <0.1% and incubatedat 37° C. in 5% CO₂ for 5 days. Proliferation of the T-cells wasevaluated by flow cytometry.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from T-cells (e.g., GM-CSF, IFNγ, andTNFα) and/or surface expression of cell surface markers on T-cells(e.g., CD69) that was indicative of T-cell activation. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance T-cellproliferation. Cbl-b inhibitors were tested for their effects on T-cellactivation in the presence of costimulation and where conditions weresuboptimal for priming.

Human T-Cell In Vitro Models of T-Cell Exhaustion

T-cell exhaustion was characterized by cells having a poor effectorresponse and a sustained level of inhibitory receptor expression thatresults in T-cell dysfunction in response to chronic infections andcancer. In vitro models of T-cell exhaustion include allogenic andautologous models. In an autologous model, myeloid cells and SEB(Staphylococcal enterotoxin B, Millipore) were used to stimulateanti-CD3 stimulated T-cells. Peripheral blood mononuclear cells (PBMC)were obtained either: 1) by using Ficoll-Paque™ (GE Healthcare) forseparation of peripheral blood hematopoietic cells from buffy coats ofhealthy human donors; or 2) directly from LeukoPak donations. Monocyteswere isolated with commercial kits using negative selection withStemcell Technologies EasySep Human Monocyte Enrichment Kit without CD16Depletion (Catalog #19058) following the manufacturer's protocol.Isolated monocytes were cultured in complete media (e.g., RPMI 1640 withno additives, 10% HI FBS, 1× Glutamine and 1× β-mercaptoethanol) with 50ng/mL recombinant human M-CSF (R&D System or Peprotech). Cells wereplated at 2×10⁶ cells per well (Day 0) and cultured for 5 days and werefed with fresh media and cytokines on day 2. On day 5 IFNγ was added at100 ng/mL and the cells were incubated overnight. Primary human T-cellsfrom the same donor were isolated from PBMCs with a commercial kit usingnegative selection (with Stemcell Technologies EasySep Human T-cellIsolation Kit (Catalog #17951) following the manufacturer's protocol.Purity was confirmed by surface marker detection by flow cytometry forCD4, CD8, CD45RA, CD45RO, CD19, CD14, CD56, and CD3 (BD Biosciences).3×10⁶ cells per/mL T-cells were stimulated with 10 μg/mL of plate boundanti-CD3 antibody (Clone UCHT-1) for 5 days. This was done in parallelwith myeloid cell generation. On day 6, 2.5×10⁴ T-cells were added perwell, 12.5×10³ myeloid cells per well and SEB antigen (0.1 μg/mL) wereadded to wells of a round bottom 96-well plate. Test agents (e.g., Cbl-binhibitor compounds) or controls (e.g., checkpoint neutralizingantibodies such as anti-PD1 antibody) were added to the wells at theindicated concentrations (e.g., 10 μM). Cells were cultured for 3 daysat which point cell free supernatants were collected and assessed forsecreted cytokines (e.g., GM-CSF, IFNγ, and IL-2) by ELISA (R&D Systems,Peprotech or Life Technologies) or Luminex multiplex kits (Procarta LifeTechnologies). The T-cells were stained for a panel of surface markersincluding checkpoint inhibitors (e.g., CTLA4) and evaluated by flowcytometry for Cbl-b inhibitor effects.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from exhausted T-cells (e.g., GM-CSF,IFNγ, and IL-2) in the presence of myeloid cells, which was indicativeof decreased T-cell exhaustion. Cbl-b inhibitors are also tested fortheir effects on checkpoint modulator expression levels followingactivation of exhausted T-cells.

Human T-Cell In Vitro Models of T-Cell Anergy

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells assessed by flow cytometry. Thecells were activated with immobilized anti-CD3 antibody (OKT3) andsoluble anti-CD28 antibody (28.2) for two days at which time they werewashed and allowed to rest for three days in the absence of stimulation.They were then treated with ionomycin (Sigma) for 18-24 hours to induceanergy. Following two washes to remove the ionomycin from the samples,Cbl-b inhibitor compounds were added to the cells at the indicatedconcentrations (e.g., 10, 1.11, and 0.37 μM) and incubated for 1 hour.The cells were then re-challenged with anti-CD3 antibody and anti-CD28antibody for 24 hours at which point cell free supernatants werecollected and assessed for cytokines (e.g., IFNγ) by ELISA (R&D Systemsor Peprotech) or Luminex multiplex kits (Procarta Life Technologies)following the manufacturer's protocols.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from anergic T-cells (e.g., IFNγ), whichwas indicative of decreased T-cell tolerance.

In Vivo Activity of Cbl-b Inhibitors

A method of determining the pharmacodynamic profile of Cbl-b inhibitorswas performed by dosing strains of mice with competent immune systemssuch as C57BL/6 or BALB/c with a Cbl-b inhibitor. The Cbl-b inhibitorwas dissolved in a suitable formulation and administered by one ofvarious routes such as intravenous (IV), intraperitoneal (IP),subcutaneous (SC), or oral (PO), at a suitable dose level and frequency(e.g., twice per day BID or thrice per day TID) as informed by priorpharmacokinetic and tolerability studies. Following administration ofthe Cbl-b inhibitor, T-cells and indirectly other immune cells (e.g.,via cytokine production) were stimulated in vivo by administration of ananti-CD3 antibody or antigen-binding fragment thereof in PBS at definedamounts such as 2 μg or 10 μg per animal by routes such as IV or IP (seeHirsh et al., J. Immunol., 1989; Ferran et al., Eur. J. Immunol., 1990).Additional study control arms included groups of mice treated with avehicle formulation alone (i.e., formulation without the Cbl-b inhibitorand anti-CD3 antibody), a formulation containing the Cbl-b inhibitoralone, a formulation containing the anti-CD3 antibody alone, PBS alone,or combinations of these agents. The level of immune activation was thenassessed by analysis of plasma cytokine levels and/or expression ofactivation markers on immune cells (e.g., T-cells). Blood or lymphoidorgans (e.g., spleen) were collected at defined time points (e.g., 8hours or 24 hours). Blood samples were processed to collect plasma fordetermination of cytokine levels using standard methods known in theart. Cytokines measured included IL-2, IFNγ, and TNFα. Additional bloodsamples and lymphoid tissues were processed for flow cytometric analysisof immune cells (e.g., T-cells) using standard methods to determineexpression of cell type-specific markers and activation markers such asCD25 and/or CD69. Augmentation of immune stimulation by Cbl-b inhibitoradministration was assessed by comparing the relative concentrations ofcytokines in plasma, or the expression levels of activation markers onimmune cells between appropriate groups (e.g., mice treated with Cbl-binhibitor and 2 μg anti-CD3 antibody versus mice treated with vehicleand 2 μg anti-CD3 antibody).

Cbl-b inhibitors were tested to determine their ability to induce orenhance the level of cytokines (e.g., IL-2, IFNγ, and TNFα) in bloodobtained from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance theexpression of cell surface markers on T-cells (e.g., CD25 and/or CD69)isolated from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response.

B-Cell Activation Assay

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Human primary B-cells were isolatedfrom the PBMCs utilizing negative selection with commercial kitsfollowing the manufacturer's protocol (Stemcell Technologies Catalog#17954) to yield>95% CD20+ cells assessed by flow cytometry. Primaryhuman B-cells were plated at 0.7-1×10⁵ per well in a 96-well plate withCbl-b inhibitors over a dose ranging from 10 μM to 1 nM and incubated at37° C. 5% CO₂, with a final DMSO concentration of <0.5%. Cells werestimulated with anti-IgM for 20 hours at 37° C. 5% CO₂. Surfaceactivation markers on mature CD20⁺ IgD⁺ B-cells were monitored by FACSusing an anti-CD69 antibody (BD Biosciences).

Cbl-b inhibitors were tested to determine their ability to induce orenhance surface expression of cell surface markers on B-cells (e.g.,CD69), which was indicative of B-cell activation.

Purification and Activation of Primary Human NK-Cells.

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary NK-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-092-657 or Stemcell Technologies Catalog #17955) to yield>92%CD56+, CD3-cells as assessed by flow cytometry. The cells were culturedovernight with IL-2 (60 ng/mL) at 37° C. 5% CO₂. Cbl-b inhibitors wereadded 1 hour prior to stimulation and incubated at 37° C. 5% CO₂ at aspecific concentration (e.g., 10 μM, 1 μM, or 0.1 μM) with a final DMSOconcentration of <0.1%. NK-cells were co-cultured with target cells thatwere engineered to have a red nucleus (K562 NucRed) measurable by flowcytometry. K562 NucRed cells were produced by transduction of K562 cellswith IncuCyte NucLight Red Lentivirus reagent (Catalog #4476) andselected for 5 days. Clonal populations were isolated and expanded usingstandard tissue culture techniques, and individual clones were validatedby comparison to wildtype K562 cells in NK-cell killing assays. Thecells were mixed at the indicated ratios (e.g., 5:1, 1:1, or 1:5) of NK(effector cells) to K562 NucRed (target cells) for 6 hours. Cell freesupernatants were collected and analyzed for cytokine secretion (e.g.,TNFα, IFNγ, or MIP1β) by ELISA or Luminex multiplex kits following themanufacturer's protocol. IFNγ secretion was assessed using an R&DSystems ELISA kit (Catalog #DY285), TNFα secretion was assessed using anR&D Systems ELISA kit (Catalog #DY210), and MIP1β secretion was assessedusing an R&D Systems ELISA kit (Catalog #DY271).

Biological Example 4: Evaluation of a Cbl-b Inhibitor in Combinationwith an Immune Checkpoint Inhibitor for Treating Cancer

Tumor microenvironments exploit T-cell inhibitory pathways as amechanism to evade anti-tumor immune responses. The use of immunecheckpoint inhibitors such as inhibitors of PD-1, PD-L1, and CTLA-4 haveresulted in strikingly efficacious and durable responses against sometumor types (Marshall and Djamgoz, Front Oncol, 8:315, 2018). However,the response to immune checkpoint inhibitor monotherapy is not universaland therefore benefits only a small subset of cancer patients (Lv etal., Journal for ImmunoTherapy of Cancer, 7:159, 2019). This exampledescribes the evaluation of a combination therapy for treating cancerincluding an immune checkpoint inhibitor and a Cbl-b inhibitor.

In brief, combination therapies were tested in strains of mice withcompetent immune systems (e.g., C57BL/6 or BALB/c) in whom syngeneictumors can be grown. Syngeneic murine tumor cells were injectedsubcutaneously: CT26 colon cancer cells in BALB/c mice; TC-1 lung cancercells in C57BL/6 mice; or MC-38 colon cancer cells in C57BL/6 mice.Tumors were allowed to grow to up to 100-200 mm³ at which time theanimals were randomized and treatment started. Alternatively, treatmentwas administered in a prophylactic setting within 1-3 days of tumor cellimplant. The Cbl-b inhibitor was dissolved in a suitable formulation andadministered at a suitable dose level and frequency as informed by priorpharmacokinetic and tolerability studies. The Cbl-b inhibitorformulation was administered orally (PO) or parenterally (e.g., IV, IP,SC, or intratumorally at one to three injection sites per tumor). Theimmune checkpoint inhibitor formulation was administered by IP injectionevery three days (e.g., days 1, 4, and 7). In addition to the test groupof mice who receive the combination therapy, the study included controlgroups of mice who received either the vehicle formulation alone, theCbl-b inhibitor formulation alone, or the immune checkpoint inhibitoralone.

The level of response was evaluated by measuring tumor growth andcomparing tumor growth in the test mice versus the control mice. Thelevel of immune activation was assessed by collecting tumors foranalysis of tumor infiltrating lymphocytes (TILs). TILs and lymphoidtissues were processed for flow cytometric analysis using standardmethods to determine cell lineage, expression of cell type-specificmarkers, and expression of activation markers such as granzyme B, PD-1,TIM3, and LAG3. Augmentation of the anti-tumor immune response by thecombination therapy was assessed by comparing the relative percentage ofimmune cell populations in the tumor, and the relative levels ofexpression of activation markers on immune cells in mice of the test andstudy groups.

Biological Example 5: Evaluation of a Cbl-b Inhibitor in Combinationwith an Anti-Neoplastic Agent for Treating Cancer

Chemotherapy has been reported to have a positive immunologic effect ontumor infiltrating lymphocytes (Lazzari et al., Ther Adv Med Oncol,10:1-12, 2018), with the balance of regulatory and effector immune cellsinfluencing prognosis. In addition, chemotherapy is contemplated toincrease the intratumoral T-cell repertoire by augmenting tumor antigenpresentation. This example describes the evaluation of a combinationtherapy for treating cancer including an anti-neoplastic agent and aCbl-b inhibitor.

In brief, combination therapies were tested in strains of mice withcompetent immune systems (e.g., C57BL/6 or BALB/c) in whom syngeneictumors can be grown. Syngeneic murine tumor cells were injectedsubcutaneously: CT26 colon cancer cells in BALB/c mice; or TC-1 lungcancer cells in C57BL/6 mice. Tumors were allowed to grow up to about120 mm³ at which time the animals were randomized and treatment started.The Cbl-b inhibitor was dissolved in a suitable formulation andadministered at a suitable dose level and frequency as informed by priorpharmacokinetic and tolerability studies. The Cbl-b inhibitorformulation was administered orally (PO) or parenterally (e.g., IV, IP,SC, or intratumorally at one to three injection sites per tumor). Theanti-neoplastic agent (e.g., gemcitabine and/or oxaliplatin) wasadministered by IP injection once every three or four days. In additionto the test group of mice who received the combination therapy, thestudy included control groups of mice who received either the vehicleformulation alone, the Cbl-b inhibitor formulation alone, or theanti-neoplastic agent alone.

The level of response was evaluated by measuring tumor growth andcomparing tumor growth in the test mice versus the control mice. Thelevel of immune activation was assessed by collecting tumors foranalysis of tumor infiltrating lymphocytes (TILs). TILs and lymphoidtissues were processed for flow cytometric analysis using standardmethods to determine cell lineage, expression of cell type-specificmarkers, and expression of activation markers such as granzyme B, PD-1,TIM3, and LAG3. Augmentation of the anti-tumor immune response by thecombination therapy was assessed by comparing the relative percentage ofimmune cell populations in the tumor, and the relative levels ofexpression of activation markers on immune cells in mice of the test andstudy groups.

Biological Example 6: Evaluation of a Cbl-b Inhibitor in Combinationwith Radiation Therapy for Treating Cancer

Ablative radiation therapy targeting local tumors limits damage tonormal tissue and has the ability to enhance the diversity of the T-cellreceptor repertoire by increasing the presence of tumor antigens (Lee etal., Blood, 114: 589-595, 2009). Radiotherapy at one site has beenreported to lead to regression of distant site tumors that were notirradiated (Ngwa et al., Nat Rev Cancer, 18: 313-322, 2018). Thesystemic effect of a localized therapy is termed an “abscopal effect”,which in the context of radiation therapy is thought to involve theimmune system. This example describes the evaluation of a combinationtherapy for treating cancer including radiation therapy and a Cbl-binhibitor.

In brief, combination therapies were tested in strains of mice withcompetent immune systems (e.g., C57BL/6 or BALB/c) in whom syngeneictumors can be grown. Syngeneic murine tumor cells were injectedsubcutaneously: CT26 colon cancer cells in BALB/c mice; or B16-F10melanoma cells in C57BL/6 mice. Tumors were allowed to grow up to about80 mm³ at which time the animals were randomized and treatment started.In some studies, tumor cells were implanted in both flanks and only onetumor was treated to assess the abscopal effect. The Cbl-b inhibitor wasdissolved in a suitable formulation and administered at a suitable doselevel and frequency as informed by prior pharmacokinetic andtolerability studies. The Cbl-b inhibitor formulation was administeredorally (PO) or parenterally (e.g., IV, IP, SC, or intratumorally at oneto three injection sites per tumor). Radiation therapy was administeredonce at a dose of 20 grays using an X-ray based focal beam irradiator.In addition to the test group of mice who received the combinationtherapy, the study included control groups of mice who received eitherthe vehicle formulation alone, the Cbl-b inhibitor formulation alone, orradiation therapy alone.

The level of response was evaluated by measuring tumor growth andcomparing tumor growth in the test mice versus the control mice. Thelevel of immune activation was assessed by collecting tumors foranalysis of tumor infiltrating lymphocytes (TILs). TILs and lymphoidtissues were processed for flow cytometric analysis using standardmethods to determine cell lineage, expression of cell type-specificmarkers, and expression of activation markers such as granzyme B, PD-1,TIM3, and LAG3. Augmentation of the anti-tumor immune response by thecombination therapy was assessed by comparing the relative percentage ofimmune cell populations in the tumor, and the relative levels ofexpression of activation markers on immune cells in mice of the test andstudy groups.

Biological Example 7: Evaluation of a Cbl-B Inhibitor in Combinationwith Adoptive Cell Therapy for Treating Cancer

Adoptive cell therapy (ACT) utilizing autologous tumor-specific T-cellsleverages the natural function of T-cells to specifically recognize andeliminate target cells (Hinrichs and Rosenberg, Immunol Rev, 257: 56-71,2014). Specificity of tumor infiltrating lymphocytes (TILs) is due totheir ability to recognize tumor-associated antigens, includingneoantigens derived from products of mutated genes. This exampledescribes the evaluation of an in vivo lympho-conditioning program witha Cbl-b inhibitor prior to ex vivo expansion of TILs for treating cancerwith ACT.

Strains of mice with competent immune systems (e.g., C57BL/6 or BALB/c)in whom syngeneic tumors can be grown were utilized. Syngeneic murinetumor cells were injected subcutaneously or intravenously: 4T1 breastcancer cells in BALB/c mice; RENCA kidney cancer cells in BALB/c mice;B16-F10 melanoma cells in C57BL/6 mice; 3LL lung cancer cells in C57BL/6mice; or MC-38 colon cancer cells in C57BL/6 mice. Tumors were allowedto grow up to about 50-600 mm³ at which time the animals were randomizedand treatment started. The Cbl-b inhibitor was dissolved in a suitableformulation and administered at a suitable dose level and frequency asinformed by prior pharmacokinetic and tolerability studies. The Cbl-binhibitor formulation was administered orally (PO) or parenterally(e.g., IV, IP, SC, or intratumorally at one to three injection sites pertumor). In addition to the test group of mice who received the Cbl-binhibitor prior to tumor harvest, a control group of mice receivedeither the vehicle formulation alone or was left untreated prior totumor harvest. Tumor tissue was harvested either from the primary tumoror from tissues with metastases (e.g., lung). The tissues were mincedand cultured in medium in the presence or absence of one or moreexogenous T-cell growth factors (e.g., IL-2, IL-7, IL-15, and/or IL-21)under conditions suitable for expansion of TILs. Expansion of TILs wasdone in the presence or absence of the Cbl-b inhibitor. Expanded TILswere assessed for phenotype by flow cytometric analysis by measuringexpression of markers for memory, effector, and stemness (e.g., CD95,TCF7, CD62L, CD44, etc.). Upon successful expansion of the TILs, tumorbearing mice were infused with TILs in the presence or absence of theCbl-b inhibitor to assess the effect of lympho-conditioning and/orsubsequent in vivo treatment on TIL engraftment and anti-tumor immuneresponses.

Anti-tumor efficacy of ACT was assessed through tumor measurements todetermine the level of tumor growth inhibition by TILs.

Biological Example 8: Evaluation of Cbl-b Inhibition by CandidateInhibitors

Candidate compounds were evaluated for their ability to bind and inhibitCbl-b, an E3 ubiquitin-protein ligase, as evidenced by their ability todisplace a fluorophore-labeled probe bound to Cbl-b.

Materials and Methods

Cbl-b Displacement Assay (Cbl-b Inhibition Assay)

The ability of candidate compounds to displace a known inhibitor andthereby inhibit Cbl-b activity was measured by monitoring theinteraction of Cbl-b with a fluorophore-labeled probe in the presence ofthe candidate compound. A truncated variant of Cbl-b (UniProt numberQ13191) containing residues 36-427 and an Avitag at its N-terminus wasco-expressed with BirA biotin ligase and purified using a standardprotocol (see Dou et al., Nature Structural and Molecular Biology 8:982-987, 2013; Avidity LLC).

Fluorescently-labeled inhibitor probe was synthesized and tagged withBODIPY FL. Cbl-b displacement assays were performed in a 384-well plateat room temperature in a 10 μl reaction volume by pre-incubating 0.5 nMCbl-b or 0.125 nM Cbl-b (final concentration, indicated as “High” and“Low”, respectively) in an assay buffer containing 20 mM HEPES pH 7.5,150 mM NaCl, 0.01% Triton X-100, 0.01% BSA and 0.5 mM TCEP in thepresence of a candidate compound in 1% DMSO (final concentration) for 1hour. After incubation in the presence of the candidate compound, theplate was incubated for an additional 1 hour in the presence of anapproximate EC₄₀ binding saturation consisting of 150 nMfluorescently-labeled inhibitor probe and 2 nM Streptavidin-Terbium(Cisbio) (final concentrations). Following the 1 hour incubation, theplates were read for TR-FRET signal at 520/620 nM using an Envisionplate reader (Perkin Elmer). The presence of a TR-FRET signal indicatedthat the probe was not displaced from Cbl-b by the compound candidate.The absence of a FRET signal indicated that the probe was displaced fromCbl-b by the compound candidate.

Results

The resulting data for the Cbl-b activity assays were analyzed usingstandard methods to determine the IC₅₀ values for the tested compounds.In Table 8-1, compounds were ranked into bins as follows for IC₅₀: Aindicates <1 nM; B indicates 1 nM-5 nM; and C indicates >5 nM.

TABLE 8-1 Cbl-b inhibition by tested compounds Cbl-b Cbl-b Compound IC₅₀IC₅₀ No. (High) (Low) 1 A 2 A 8 A 28 B 32 A 34 B 39 A 44 A 53 C 54 C 55B 56 C 57 C 58 C 59 B 60 B 61 B Blank cell indicates data not available.

Biological Example 9: Evaluation of T-cell Activation by Cbl-bInhibitors

Cbl-b inhibitors were evaluated for their ability to activate T-cells.

Materials and Methods

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.The cells were rested overnight at 37° C. 5% CO₂. The Cbl-inhibitor wasadded to 1×10⁵ cells per well and the plate was incubated for one hourat 37° C. in 5% CO₂ at the concentrations indicated (Table 9-1) with afinal DMSO concentration of <0.1%. For samples stimulated with anti-CD3antibody and anti-CD28 antibody (anti-CD3/anti-CD28), the Cbl-binhibitor concentrations tested were 1 μM, and 0.3 μM. For samplesstimulated with anti-CD3 antibody alone (anti-CD3), the Cbl-b inhibitorconcentrations tested were 3 μM, and 1 μM. Following incubation with theCbl-b inhibitor, primary human total T-cells were stimulated with eitherplate bound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3antibody (OKT3) with soluble anti-CD28 antibody (28.2) (LifeTechnologies). To prepare plates with plate bound anti-CD3 antibody(OKT3), 96-well round bottom tissue culture plates were coated with 100μL of anti-CD3 antibody (OKT3) at 10 pg/mL for 4 hours at 37° C. 5% CO₂in phosphate buffered saline (PBS). The plates were washed with PBS onceprior to adding the cells with or without soluble anti-CD28 antibody(28.2) to each well at a final concentration of 5 μg/mL. Cells werestimulated for 48 hours prior to harvesting the cell free supernatantand staining the cell population for surface marker assessment by flowcytometry. Supernatants were analyzed for cytokine secretion, includingIL-2 by ELISA (R&D Systems, Peprotech or Life Technologies) or Luminexmultiplex kits (Procarta Life Technologies) following the manufacturer'sprotocol. Cells were stained with anti-CD25 antibody (BD Biosciences) toassess levels of surface marker of activation.

Results

Readouts were reported as fold change over baseline. Baseline for thisstudy was the measurement obtained from total human T-cells stimulatedwith anti-CD3 antibody and with soluble anti-CD28 antibody, wherein thecells were not incubated with a Cbl-b inhibitor (Table 9-1). For T-cellsstimulated with anti-CD3/anti-CD28, changes greater than 2.5-fold overbaseline for IL-2 secretion and greater than 1.3-fold over baseline forCD25 surface staining were considered significant and a positiveresponse. For T-cells stimulated with anti-CD3 alone, changes greaterthan 0.1-fold over baseline for IL-2 secretion and greater than 0.6-foldover baseline for CD25 surface staining were considered significant anda positive response.

Compounds were ranked into bins according to their readouts as follows:

For IL-2 secretion with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates <10 fold, B indicates 10-15fold, A indicates >15 fold;For IL-2 secretion with anti-CD3 antibody stimulation the bins are: Cindicates ≤0.33 fold, B indicates 0.34-0.66 fold, A indicates >0.66fold;For CD25 staining with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates≤1.24 fold, B indicates1.25-1.39 fold, A indicates >1.39 fold;For CD25 staining with anti-CD3 antibody stimulation the bins are: Cindicates ≤1.04 fold, B indicates 1.05-1.14 fold, and A indicates >1.14fold.

TABLE 9-1 T-cell activation as assessed by IL-2 secretion and CD25surface staining as a consequence of stimulation with anti-CD3, oranti-CD3 and anti-CD28 IL-2 secretion IL-2 secretion CD25 staining CD25staining Cmpd CD3/CD28 CD3 CD3/CD28 CD3 No. 1 μM 0.3 μM 3 μM 1 μM 1 μM0.3 μM 3 μM l μM 1 A A A A A A A A 2 B B A B C C B B 8 A A A A A A A A28 B B B B C B C B 32 A A A A A A A A 39 A A A A A A A A 44 C C B C B BB B 53 B C B B A A A A 54 C C C B B A 55 A A B B A A A A 56 B B C A B A57 B C C A B A 58 C C C B C C 59 A A B C A B A A 60 A A B B A A A A 61 AA B B A A A A

Cbl-b inhibitors enhanced IL-2 secretion by T-cells stimulated with ananti-CD3 antibody alone or in combination with an anti-CD28 antibody.Expression of the CD25 activation marker on the surface of T cellsincreased when stimulated with an anti-CD3 antibody alone or incombination with an anti-CD28 antibody. These results indicate theidentified Cbl-b inhibitors have the ability to activate T-cells andthat such activation did not require co-stimulation with an anti-CD28antibody.

Biological Example 10: Evaluation of Immunomodulatory Effects of Cbl-bInhibitors

Cbl-b inhibitors identified from screening assays demonstrated theability to activate total human T-cells in vitro as evidenced byenhanced IL-2 secretion and expression of the CD25 surface activationmarker.

Further in vitro studies were conducted to assess additional cytokinesecretion by T-cells and expression of surface activation markers onT-cells. Additional immunomodulatory effects on T-cells contacted withthe Cbl-b inhibitors described herein were assessed, such as the abilityof a Cbl-b inhibitor to increase T-cell proliferation, decrease T-cellexhaustion, and decrease T-cell anergy. The ability of Cbl-b inhibitors,such as those described herein, to activate T-cells in vivo was alsoassessed. Other immunomodulatory effects by the Cbl-b inhibitors wereassessed, such as the ability of Cbl-b inhibitors to activate B-cellsand NK-cells.

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.For measurement of cell proliferation, cells were labeled with CellTrace Violet (Invitrogen) following the manufacturer's protocol prior toactivation by stimulation with anti-CD3 antibody alone or in combinationwith anti-CD28 antibody. The Cbl-b inhibitor was added to 1×10⁵ cellsper well at multiple concentrations (e.g., 10 μM, 1.11 μM, or 0.123 μM)with a final DMSO concentration of <0.1%. The plate was incubated forone hour at 37° C. in 5% CO₂. Following incubation with the Cbl-binhibitor, primary human total T-cells were stimulated with either platebound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3 antibody(OKT3) with soluble anti-CD28 antibody (28.2) (Life Technologies). Toprepare plates with plate bound anti-CD3 antibody (OKT3), 96-well roundbottom tissue culture plates were coated with 100 μL of anti-CD3antibody (OKT3) at 10 μg/mL for 4 hours at 37° C. 5% CO₂ in phosphatebuffered saline (PBS). The plates were washed with PBS prior to addingthe cells with or without soluble anti-CD28 antibody (28.2) to each wellat a final concentration of 5 μg/mL. Cells were stimulated for 48 hoursprior to harvesting the cell free supernatant and staining the cellpopulation for surface marker assessment by flow cytometry. Supernatantswere analyzed for cytokine secretion (e.g., GM-CSF, IFNγ, and TNFα) byELISA (R&D Systems, Peprotech or Life Technologies) or Luminex multiplexkits (Procarta Life Technologies) following the manufacturer's protocol.Cells were stained with anti-CD69 (BD Biosciences) to assess levels ofsurface markers of activation. Proliferation was measured by flowcytometry and data was analyzed with FlowJo v7.6.5 or v10. Readouts werereported as fold change over baseline. In some embodiments, baseline wasthe measurement obtained from total human T-cells stimulated withanti-CD3 antibody alone, wherein the cells were not incubated with aCbl-b inhibitor. In some embodiments, baseline was the measurementobtained from total human T-cells stimulated with anti-CD3 antibody andanti-CD28 antibody, where the cells were not incubated with a Cbl-binhibitor.

Cbl-b inhibitor effects on primary human T-cells were also evaluated inthe context of an allogenic mixed lymphocyte reaction (MLR). Allogenicimmature dendritic cells were generated under the following conditions.Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Monocytes were isolated from the PMBCsutilizing positive selection with a commercial kit following themanufacturer's protocol (StemCells Catalog #17858) to yield>95% CD14+cells as assessed by flow cytometry. Monocytes were cultured with 30ng/mL of recombinant human GM-CSF and 20 ng/mL of recombinant human IL-4for seven days to generate immature dendritic cells. Monocytes andT-cells were either isolated fresh from peripheral blood or thawed fromfrozen stocks. Human T-cells were isolated, labeled with CFSE andincubated with inhibitors as described above. The Cbl-b inhibitor wasadded to 1×10⁵ T-cells in coculture with 2×10³ allogenic immaturedendritic cells per well at multiple concentrations (e.g., 10 μM, or1.11 μM) with a final DMSO concentration of <0.1% and incubated at 37°C. in 5% CO₂ for 5 days. Proliferation of the T-cells was evaluated byflow cytometry.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from T-cells (e.g., GM-CSF, IFNγ, andTNFα) and/or surface expression of cell surface markers on T-cells(e.g., CD69) that was indicative of T-cell activation. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance T-cellproliferation. Cbl-b inhibitors were tested for their effects on T-cellactivation in the presence of costimulation and where conditions aresuboptimal for priming.

Human T-Cell In Vitro Models of T-Cell Exhaustion

T-cell exhaustion was characterized by cells having a poor effectorresponse and a sustained level of inhibitory receptor expression thatresults in T-cell dysfunction in response to chronic infections andcancer. In vitro models of T-cell exhaustion include allogenic andautologous models. In an autologous model, myeloid cells and SEB(Staphylococcal enterotoxin B, Millipore) were used to stimulateanti-CD3 stimulated T-cells. Peripheral blood mononuclear cells (PBMC)were obtained either: 1) by using Ficoll-Paque™ (GE Healthcare) forseparation of peripheral blood hematopoietic cells from buffy coats ofhealthy human donors; or 2) directly from LeukoPak donations. Monocyteswere isolated with commercial kits using negative selection withStemCells EasySep Human Monocyte Enrichment Kit without CD16 Depletion(Catalog #19058) following the manufacturer's protocol. Isolatedmonocytes were cultured in complete media (e.g. RPMI 1640 with noadditives, 10% HI FBS, 1× Glutamine and 1× β-mercaptoethanol) with 50ng/mL recombinant human M-CSF (R&D System or Peprotech). Cells wereplated at 2×10⁶ cells per well (Day 0) and cultured for 5 days and werefed with fresh media and cytokines on day 2. On day 5, IFNγ is added at100 ng/mL and the cells were incubated overnight. Primary human T-cellsfrom the same donor were isolated from PBMCs with a commercial kit usingnegative selection (with Stemcell Technologies EasySep Human T-cellIsolation Kit (Catalog #17951) following the manufacturer's protocol.Purity was confirmed by surface marker detection by flow cytometry forCD4, CD8, CD45RA, CD45RO, CD19, CD14, CD56, and CD3 (BD Biosciences).3×10⁶ cells per/mL T-cells were stimulated with 10 μg/mL of plate boundanti-CD3 antibody (Clone UCHT-1) for 5 days. This was done in parallelwith myeloid cell generation. On day 6, 2.5×10⁴ T-cells were added perwell, 12.5×10³ myeloid cells per well and SEB antigen (0.1 μg/mL) wereadded to wells of a round bottom 96-well plate. Test agents (e.g. Cbl-binhibitor compounds) or controls (e.g., checkpoint neutralizingantibodies such as anti-PD1 antibody) were added to the wells at theindicated concentrations (e.g., 10 μM). Cells were cultured for 3 daysat which point cell free supernatants were collected and assessed forsecreted cytokines (e.g., GM-CSF, IFNγ, and IL-2) by ELISA (R&D Systems,Peprotech or Life Technologies) or Luminex multiplex kits (Procarta LifeTechnologies). The T-cells were stained for a panel of surface markersincluding checkpoint inhibitors (e.g., CTLA4) and evaluated by flowcytometry for Cbl-b inhibitor effects.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from exhausted T-cells (e.g., GM-CSF,IFNγ, and IL-2) in the presence of myeloid cells, which was indicativeof decreased T-cell exhaustion. Cbl-b inhibitors were also tested fortheir effects on checkpoint modulator expression levels followingactivation of exhausted T-cells.

Human T-Cell In Vitro Models of T-Cell Anergy

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells assessed by flow cytometry. Thecells were activated with immobilized anti-CD3 antibody (OKT3) andsoluble anti-CD28 antibody (28.2) for two days at which time they werewashed and allowed to rest for three days in the absence of stimulation.They were then treated with ionomycin (Sigma) for 18-24 hours to induceanergy. Following two washes to remove the ionomycin from the samples,Cbl-b inhibitor compounds were added to the cells at the indicatedconcentrations (e.g., 10, 1.11, and 0.37 μM) and incubated for 1 hour.The cells were then re-challenged with anti-CD3 antibody and anti-CD28antibody for 24 hours at which point cell free supernatants werecollected and assessed for cytokines (e.g., IFNγ) by ELISA (R&D Systemsor Peprotech) or Luminex multiplex kits (Procarta Life Technologies)following the manufacturer's protocols.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from anergic T-cells (e.g., IFNγ), whichwas indicative of decreased T-cell tolerance.

In Vivo Activity of Cbl-b Inhibitors

A method of determining the pharmacodynamic profile of Cbl-b inhibitorswas performed by dosing strains of mice with competent immune systemssuch as C57BL/6 or BALB/c with a Cbl-b inhibitor. The Cbl-b inhibitorwas dissolved in a suitable formulation and administered by one ofvarious routes such as intravenous (IV), intraperitoneal (IP),subcutaneous (SC), or oral (PO), at a suitable dose level and frequency(e.g., twice per day BID or thrice per day TID) as informed by priorpharmacokinetic and tolerability studies. Following administration ofthe Cbl-b inhibitor, T-cells and indirectly other immune cells (e.g.,via cytokine production) were stimulated in vivo by administration of ananti-CD3 antibody or antigen-binding fragment thereof in PBS at definedamounts such as 2 μg or 10 μg per animal by routes such as IV or IP (seeHirsh et al., J. Immunol., 1989; Ferran, et al., Eur. J. Immunol.,1990). Additional study control arms included groups of mice treatedwith a vehicle formulation alone (i.e., formulation without the Cbl-binhibitor and anti-CD3 antibody), a formulation containing the Cbl-binhibitor alone, a formulation containing the anti-CD3 antibody alone,PBS alone, or combinations of these agents. The level of immuneactivation was then assessed by analysis of plasma cytokine levelsand/or expression of activation markers on immune cells (e.g., T-cells).Blood or lymphoid organs (e.g., spleen) were collected at defined timepoints (e.g., 8 hours or 24 hours). Blood samples were processed tocollect plasma for determination of cytokine levels using standardmethods known in the art. Cytokines measured included IL-2, IFNγ, andTNFα. Additional blood samples and lymphoid tissues were processed forflow cytometric analysis of immune cells (e.g., T-cells) using standardmethods to determine expression of cell type-specific markers andactivation markers such as CD25 and/or CD69. Augmentation of immunestimulation by Cbl-b inhibitor administration was assessed by comparingthe relative concentrations of cytokines in plasma, or the expressionlevels of activation markers on immune cells between appropriate groups(e.g., mice treated with Cbl-b inhibitor and 2 μg anti-CD3 antibodyversus mice treated with vehicle and 2 μg anti-CD3 antibody).

Cbl-b inhibitors were tested to determine their ability to induce orenhance the level of cytokines (e.g., IL-2, IFNγ, and TNFα) in bloodobtained from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance theexpression of cell surface markers on T-cells (e.g., CD25 and/or CD69)isolated from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response.

B-Cell Activation Assay

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Human primary B-cells were isolatedfrom the PBMCs utilizing negative selection with commercial kitsfollowing the manufacturer's protocol (Stemcell Technologies Catalog#17954) to yield>95% CD20+ cells assessed by flow cytometry. Primaryhuman B-cells were plated at 0.7-1×10⁵ per well in a 96-well plate withCbl-b inhibitors over a dose ranging from 10 μM to 1 nM and incubated at37° C. 5% CO₂, with a final DMSO concentration of <0.5%. Cells werestimulated with anti-IgM for 20 hours at 37° C. 5% CO₂. Surfaceactivation markers on mature CD20⁺ IgD⁺ B-cells were monitored by FACSusing an anti-CD69 antibody (BD Biosciences).

Cbl-b inhibitors were tested to determine their ability to induce orenhance surface expression of cell surface markers on B-cells (e.g.,CD69), which was indicative of B-cell activation.

Purification and Activation of Primary Human NK-Cells.

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary NK-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-092-657 or Stemcell Technologies Catalog #17955) to yield>92%CD56+, CD3-cells as assessed by flow cytometry. The cells were culturedovernight with IL-2 (60 ng/mL) at 37° C. 5% CO₂. Cbl-b inhibitors wereadded 1 hour prior to stimulation and incubated at 37° C. 5% CO₂ at aspecific concentration (e.g., 10 μM, 1 μM, or 0.1 μM) with a final DMSOconcentration of <0.1%. NK-cells were co-cultured with target cells thatwere engineered to have a red nucleus (K562 NucRed) measurable by flowcytometry. K562 NucRed cells were produced by transduction of K562 cellswith IncuCyte NucLight Red Lentivirus reagent (Catalog #4476) andselected for 5 days. Clonal populations were isolated and expanded usingstandard tissue culture techniques, and individual clones were validatedby comparison to wildtype K562 cells in NK-cell killing assays. Thecells were mixed at the indicated ratios (e.g., 5:1, 1:1, or 1:5) of NK(effector cells) to K562 NucRed (target cells) for 6 hours. Cell freesupernatants were collected and analyzed for cytokine secretion (e.g.,TNFα, IFNγ, or MIP1β) by ELISA or Luminex multiplex kits following themanufacturer's protocol. IFNγ secretion was assessed using an R&DSystems ELISA kit (Catalog #DY285), TNFα secretion was assessed using anR&D Systems ELISA kit (Catalog #DY210), and MIP1β secretion was assessedusing an R&D Systems ELISA kit (Catalog #DY271).

Biological Example 11: Evaluation of a Cbl-b Inhibitor in Combinationwith a Cancer Vaccine for Treating Cancer

This example describes the evaluation of a combination therapy fortreating cancer in mice including a cancer vaccine and a Cbl-binhibitor.

Materials and Methods

In brief, the combination therapy was tested in C57BL/6 mice bearing asyngeneic TC-1 cell tumor. TC-1 cells were derived from primary murinelung cells, which had been transfected with human papillomavirus strain16 (HPV16) early proteins 6 and 7 (E6 and E7) and an activated humanc-Ha-ras oncogene (Lin et al., Cancer Research, 56: 21-26, 1996). Either2.5×10⁴ (first study) or 5.0×10⁴ (second study) TC-1 cells (obtainedfrom Johns Hopkins University) were injected subcutaneously into the midflank of each mouse. Tumors were allowed to grow to 50-100 mm³ at whichtime the animals were randomized and treatment was begun (day 0).

The HPV16 E7 cancer vaccine (Shrimali et al., Cancer Immunol Res, 5:755-766, 2017) included 100 μg of a synthetic peptide with the aminoacid sequence of a CTL epitope of residues 49-57 of E7 (RAHYNIVTF setforth as SEQ ID NO:2), mixed with 20 μg of a synthetic peptide with theamino acid sequence of a Th epitope known as PADRE (aK-Cha-VAAWTLKAAaset for as SEQ ID NO:3, where “a” is D alanine and “Cha” isL-cyclohexylalanine) and 20 μg of a Quil-A® adjuvant marketed byBrenntag Biosector A/S, which contains purified quillaja saponins. TheHPV16 E7 vaccine or Dulbecco's phosphate-buffered saline (DPBS) wasadministered subcutaneously (SC) on days 2 and 9 (=day 16 and day 23post TC-1 injection). The Cbl-b inhibitor was dissolved in a suitableformulation and administered at a suitable dose level and frequency asinformed by prior pharmacokinetic and tolerability studies. In initialstudies, the Cbl-b inhibitor (Compound No. 53) in a suitable vehicle, orthe vehicle, was administered orally (PO) at a dose of 180 mg/kg twiceper day (BID) beginning on day 0 (=day 14 post TC-1 injection). Infurther studies, a Cbl-b inhibitor formulation was administered orally(PO) or parenterally (e.g., IV, IP, SC, or intratumorally at one tothree injection sites per tumor). In addition to the test group of micewho received the combination therapy (the Cbl-b inhibitor formulationplus the HPV16 E7 vaccine), the study included control groups of micewho received: the vehicle formulation plus DPBS, the Cbl-b inhibitorformulation plus DPBS, or the vehicle formulation plus the HPV16 E7vaccine.

Tumor size was recorded twice a week starting on day 0 (day 14 post TC-1injection) and continued through duration of dosing until humaneendpoints were reached between day 33 and day 42 (=between day 47 andday 56 post TC-1 injection). The level of response was evaluated bycomparing tumor growth in the test mice versus the control mice (10 miceper group). In addition, the level of immune activation was assessed bycollecting tumors for analysis of tumor infiltrating lymphocytes (TILs)on day 10 (=day 24 post TC-1 injection) in test and control mice (6 miceper group). TILs were processed for flow cytometric analysis usingstandard methods to determine antigen specificity, expression of celltype-specific markers, and expression of activation markers such asgranzyme B, CD25, CD69, PD-1, and LAG3. In further experiments, PBMCsand/or splenocytes were also collected and processed. Augmentation ofthe anti-tumor immune response by the combination therapy was assessedby comparing the relative percentage of total or E7-specific (tumorantigen-specific) CD8+ T cells, and the relative levels of expression ofactivation markers on immune cells in mice of the test and study groups.

Results

As shown in Table 11-1, Cbl-b inhibition in combination with tumorpeptide vaccination promoted tumor infiltration by tumorpeptide-specific CD8+ T cells. In addition, the combination therapyincluding a Cbl-b inhibitor and a tumor peptide vaccine resulted inelevated levels in tumors of both tumor peptide-specific (E7+) and other(E7-) activated CD8+ T cells. Moreover, the combination therapyincluding a Cbl-b inhibitor and a tumor peptide vaccine yielded morepartial responders (6/10 mice) than did the respective monotherapies(0/10 partial responders to the Cbl-b inhibitor alone and 3/10 partialresponders to the tumor peptide vaccine alone). In this experiment,partial responders were defined by a significant decrease in tumor size(tumor volume) relative to the size of tumors of control mice who didnot receive the Cbl-b inhibitor or the tumor peptide vaccine.

TABLE 11-1 Mean Increase in CD8+ T cells in TILs of Recipients of anHPV16 E7 Vaccine and a Cbl-b Inhibitor{circumflex over ( )} HPV16 E7Vaccine + TIL Profile Compound No. 53 % E7 positive T cells 33.7 %Granzyme positive (E7+ T cells) 7.86 % PD1 positive (E7+ T cells) 14.97% LAG3 positive (E7+ T cells) 13.49 % Granzyme positive (E7− T cells)4.12 % PD1 positive (E7− T cells) 20.23 % LAG3 positive (E7− cells) 0.96{circumflex over ( )} Percent increase is relative to CD8+ T cells inTILs of Recipients of an HPV16 E7 vaccine in the absence of a Cbl-binhibitor.

Biological Example 12: Evaluation of Cbl-b Inhibition by CandidateInhibitors

Candidate compounds were evaluated for their ability to bind and inhibitCbl-b, an E3 ubiquitin-protein ligase, as evidenced by their ability todisplace a fluorophore-labeled probe bound to Cbl-b.

Materials and Methods

Cbl-b Displacement Assay (Cbl-b Inhibition Assay)

The ability of candidate compounds to displace a known inhibitor andthereby inhibit Cbl-b activity was measured by monitoring theinteraction of Cbl-b with a fluorophore-labeled probe in the presence ofthe candidate compound. A truncated variant of Cbl-b (UniProt numberQ13191) containing residues 36-427 and an Avitag at its N-terminus wasco-expressed with BirA biotin ligase and purified using a standardprotocol (see Dou et al., Nature Structural and Molecular Biology 8:982-987, 2013; Avidity LLC).

Fluorescently-labeled inhibitor probe was synthesized and tagged withBODIPY FL. Cbl-b displacement assays were performed in a 384-well plateat room temperature in a 10 μl reaction volume by pre-incubating 0.5 nMCbl-b or 0.125 nM Cbl-b (final concentration, indicated as “High” and“Low”, respectively) in an assay buffer containing 20 mM HEPES pH 7.5,150 mM NaCl, 0.01% Triton X-100, 0.01% BSA and 0.5 mM TCEP in thepresence of a candidate compound in 1% DMSO (final concentration) for 1hour. After incubation in the presence of the candidate compound, theplate was incubated for an additional 1 hour in the presence of anapproximate EC₄₀ binding saturation consisting of 150 nMfluorescently-labeled inhibitor probe and 2 nM Streptavidin-Terbium(Cisbio) (final concentrations). Following the 1 hour incubation, theplates were read for TR-FRET signal at 520/620 nM using an Envisionplate reader (Perkin Elmer). The presence of a TR-FRET signal indicatedthat the probe was not displaced from Cbl-b by the compound candidate.The absence of a FRET signal indicated that the probe was displaced fromCbl-b by the compound candidate.

Results

The resulting data for the Cbl-b activity assays were analyzed usingstandard methods to determine the IC₅₀ values for the tested compounds.In Table 12-1, compounds were ranked into bins as follows for IC₅₀: Aindicates <1 nM; and B indicates 1 nM-5 nM; and C indicates >5 nM.

TABLE 12-1 Cbl-b inhibition by tested compounds Cbl-b Cbl-b CompoundIC₅₀ IC₅₀ No. (High) (Low) 1 A 2 A 8 A 28 B 32 A 34 B 39 A 44 A 53 C 54C 55 B 56 C 57 C 58 C 59 B 60 B 61 B Blank cell indicates data notavailable.

Biological Example 13: Evaluation of T-cell Activation by Cbl-bInhibitors

Cbl-b inhibitors were evaluated for their ability to activate T-cells.

Materials and Methods

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.The cells were rested overnight at 37° C. 5% Co₂. The Cbl-inhibitor wasadded to 1×10⁵ cells per well and the plate was incubated for one hourat 37° C. in 5% CO₂ at the concentrations indicated (Table 13-1) with afinal DMSO concentration of <0.1%. For samples stimulated with anti-CD3antibody and anti-CD28 antibody (anti-CD3/anti-CD28), the Cbl-binhibitor concentrations tested were 1 μM, and 0.3 μM. For samplesstimulated with anti-CD3 antibody alone (anti-CD3), the Cbl-b inhibitorconcentrations tested were 3 μM, and 1 μM. Following incubation with theCbl-b inhibitor, primary human total T-cells were stimulated with eitherplate bound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3antibody (OKT3) with soluble anti-CD28 antibody (28.2) (LifeTechnologies). To prepare plates with plate bound anti-CD3 antibody(OKT3), 96-well round bottom tissue culture plates were coated with 100μL of anti-CD3 antibody (OKT3) at 10 μg/mL for 4 hours at 37° C. 5% CO₂in phosphate buffered saline (PBS). The plates were washed with PBS onceprior to adding the cells with or without soluble anti-CD28 antibody(28.2) to each well at a final concentration of 5 μg/mL. Cells werestimulated for 48 hours prior to harvesting the cell free supernatantand staining the cell population for surface marker assessment by flowcytometry. Supernatants were analyzed for cytokine secretion, includingIL-2 by ELISA (R&D Systems, Peprotech or Life Technologies) or Luminexmultiplex kits (Procarta Life Technologies) following the manufacturer'sprotocol. Cells were stained with anti-CD25 antibody (BD Biosciences) toassess levels of surface marker of activation.

Results

Readouts were reported as fold change over baseline. Baseline for thisstudy was the measurement obtained from total human T-cells stimulatedwith anti-CD3 antibody and with soluble anti-CD28 antibody, wherein thecells were not incubated with a Cbl-b inhibitor (Table 13-1). ForT-cells stimulated with anti-CD3/anti-CD28, changes greater than2.5-fold over baseline for IL-2 secretion and greater than 1.3-fold overbaseline for CD25 surface staining were considered significant and apositive response. For T-cells stimulated with anti-CD3 alone, changesgreater than 0.1-fold over baseline for IL-2 secretion and greater than0.6-fold over baseline for CD25 surface staining were consideredsignificant and a positive response.

Compounds were ranked into bins according to their readouts as follows:

For IL-2 secretion with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates ≤10 fold, B indicates 11-15fold, A indicates >15 fold;For IL-2 secretion with anti-CD3 antibody stimulation the bins are: Cindicates ≤0.33 fold, B indicates 0.34-0.66 fold, A indicates >0.66fold;For CD25 staining with anti-CD3 antibody and anti-CD28 antibodyco-stimulation the bins are: C indicates ≤1.24 fold, B indicates1.25-1.39 fold, A indicates >1.39 fold;For CD25 staining with anti-CD3 antibody stimulation the bins are: Cindicates ≤1.04 fold, B indicates 1.05-1.14 fold, and A indicates >1.14fold.

TABLE 13-1 T-cell activation as assessed by IL-2 secretion and CD25surface staining as a consequence of stimulation with anti-CD3, oranti-CD3 and anti-CD28 IL-2 secretion IL-2 secretion CD25 staining CD25staining Cmpd CD3/CD28 CD3 CD3/CD28 CD3 No. 1 μM 0.3 μM 3 μM 1 μM 1 μM0.3 μM 3 μM l μM 1 A A A A A A A A 2 B B A B C C B B 8 A A A A A A A A28 B B B B C B C B 32 A A A A A A A A 39 A A A A A A A A 44 C C B C B BB B 53 B C B B A A A A 54 C C C B B A 55 A A B B A A A A 56 B B C A B A57 B C C A B A 58 C C C B C C 59 A A B C A B A A 60 A A B B A A A A 61 AA B B A A A A

Cbl-b inhibitors enhanced IL-2 secretion by T-cells stimulated with ananti-CD3 antibody alone or in combination with an anti-CD28 antibody.Expression of the CD25 activation marker on the surface of T cellsincreased when stimulated with an anti-CD3 antibody alone or incombination with an anti-CD28 antibody. These results indicate theidentified Cbl-b inhibitors have the ability to activate T-cells andthat such activation did not require co-stimulation with an anti-CD28antibody.

Biological Example 14: Evaluation of Immunomodulatory Effects of Cbl-bInhibitors

Cbl-b inhibitors identified from screening assays demonstrated theability to activate total human T-cells in vitro as evidenced byenhanced IL-2 secretion and expression of the CD25 surface activationmarker.

Further in vitro studies were conducted to assess additional cytokinesecretion by T-cells and expression of surface activation markers onT-cells. Additional immunomodulatory effects on T-cells contacted withthe Cbl-b inhibitors described herein were assessed, such as the abilityof a Cbl-b inhibitor to increase T-cell proliferation, decrease T-cellexhaustion, and decrease T-cell anergy. The ability of Cbl-b inhibitors,such as those described herein, to activate T-cells in vivo was alsoassessed. Other immunomodulatory effects by the Cbl-b inhibitors wereassessed, such as the ability of Cbl-b inhibitors to activate B-cellsand NK-cells.

Purification and Assessment of Primary Human Total T-Cell Activation

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PMBCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells as assessed by flow cytometry.For measurement of cell proliferation, cells were labeled with CellTrace Violet (Invitrogen) following the manufacturer's protocol prior toactivation by stimulation with anti-CD3 antibody alone or in combinationwith anti-CD28 antibody. The Cbl-b inhibitor was added to 1×10⁵ cellsper well at multiple concentrations (e.g., 10 μM, 1.11 μM, or 0.123 μM)with a final DMSO concentration of <0.1%. The plate was incubated forone hour at 37° C. in 5% CO₂. Following incubation with the Cbl-binhibitor, primary human total T-cells were stimulated with either platebound anti-CD3 antibody (OKT3) alone or plate bound anti-CD3 antibody(OKT3) with soluble anti-CD28 antibody (28.2) (Life Technologies). Toprepare plates with plate bound anti-CD3 antibody (OKT3), 96-well roundbottom tissue culture plates were coated with 100 μL of anti-CD3antibody (OKT3) at 10 μg/mL for 4 hours at 37° C. 5% CO₂ in phosphatebuffered saline (PBS). The plates were washed with PBS prior to addingthe cells with or without soluble anti-CD28 antibody (28.2) to each wellat a final concentration of 5 μg/mL. Cells were stimulated for 48 hoursprior to harvesting the cell free supernatant and staining the cellpopulation for surface marker assessment by flow cytometry. Supernatantswere analyzed for cytokine secretion (e.g., GM-CSF, IFNγ, and TNFα) byELISA (R&D Systems, Peprotech or Life Technologies) or Luminex multiplexkits (Procarta Life Technologies) following the manufacturer's protocol.Cells were stained with anti-CD69 (BD Biosciences) to assess levels ofsurface markers of activation. Proliferation was measured by flowcytometry and data was analyzed with FlowJo v7.6.5 or v10. Readouts werereported as fold change over baseline. In some embodiments, baseline wasthe measurement obtained from total human T-cells stimulated withanti-CD3 antibody alone, wherein the cells were not incubated with aCbl-b inhibitor. In some embodiments, baseline was the measurementobtained from total human T-cells stimulated with anti-CD3 antibody andanti-CD28 antibody, where the cells were not incubated with a Cbl-binhibitor.

Cbl-b inhibitor effects on primary human T-cells were also evaluated inthe context of an allogenic mixed lymphocyte reaction (MLR). Allogenicimmature dendritic cells were generated under the following conditions.Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Monocytes were isolated from the PMBCsutilizing positive selection with a commercial kit following themanufacturer's protocol (StemCells Catalog #17858) to yield>95% CD14+cells as assessed by flow cytometry. Monocytes were cultured with 30ng/mL of recombinant human GM-CSF and 20 ng/mL of recombinant human IL-4for seven days to generate immature dendritic cells. Monocytes andT-cells were either isolated fresh from peripheral blood or thawed fromfrozen stocks. Human T-cells were isolated, labeled with CFSE andincubated with inhibitors as described above. The Cbl-b inhibitor wasadded to 1×10⁵ T-cells in coculture with 2×10³ allogenic immaturedendritic cells per well at multiple concentrations (e.g., 10 μM, or1.11 μM) with a final DMSO concentration of <0.1% and incubated at 37°C. in 5% CO₂ for 5 days. Proliferation of the T-cells was evaluated byflow cytometry.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from T-cells (e.g., GM-CSF, IFNγ, andTNFα) and/or surface expression of cell surface markers on T-cells(e.g., CD69) that was indicative of T-cell activation. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance T-cellproliferation. Cbl-b inhibitors were tested for their effects on T-cellactivation in the presence of costimulation and where conditions weresuboptimal for priming.

Human T-Cell In Vitro Models of T-Cell Exhaustion

T-cell exhaustion was characterized by cells having a poor effectorresponse and a sustained level of inhibitory receptor expression thatresults in T-cell dysfunction in response to chronic infections andcancer. In vitro models of T-cell exhaustion include allogenic andautologous models. In an autologous model, myeloid cells and SEB(Staphylococcal enterotoxin B, Millipore) were used to stimulateanti-CD3 stimulated T-cells. Peripheral blood mononuclear cells (PBMC)were obtained either: 1) by using Ficoll-Paque™ (GE Healthcare) forseparation of peripheral blood hematopoietic cells from buffy coats ofhealthy human donors; or 2) directly from LeukoPak donations. Monocyteswere isolated with commercial kits using negative selection withStemCells EasySep Human Monocyte Enrichment Kit without CD16 Depletion(Catalog #19058) following the manufacturer's protocol. Isolatedmonocytes were cultured in complete media (e.g. RPMI 1640 with noadditives, 10% HI FBS, 1× Glutamine and 1× β-mercaptoethanol) with 50ng/mL recombinant human M-CSF (R&D System or Peprotech). Cells wereplated at 2×10⁶ cells per well (Day 0) and cultured for 5 days and werefed with fresh media and cytokines on day 2. On day 5, IFNγ was added at100 ng/mL and the cells were incubated overnight. Primary human T-cellsfrom the same donor were isolated from PBMCs with a commercial kit usingnegative selection (with Stemcell Technologies EasySep Human T-cellIsolation Kit (Catalog #17951) following the manufacturer's protocol.Purity was confirmed by surface marker detection by flow cytometry forCD4, CD8, CD45RA, CD45RO, CD19, CD14, CD56, and CD3 (BD Biosciences).3×10⁶ cells per/mL T-cells were stimulated with 10 μg/mL of plate boundanti-CD3 antibody (Clone UCHT-1) for 5 days. This was done in parallelwith myeloid cell generation. On day 6, 2.5×10⁴ T-cells were added perwell, 12.5×10³ myeloid cells per well and SEB antigen (0.1 μg/mL) wereadded to wells of a round bottom 96-well plate. Test agents (e.g. Cbl-binhibitor compounds) or controls (e.g., checkpoint neutralizingantibodies such as anti-PD1 antibody) were added to the wells at theindicated concentrations (e.g., 10 μM). Cells were cultured for 3 daysat which point cell free supernatants were collected and assessed forsecreted cytokines (e.g., GM-CSF, IFNγ, and IL-2) by ELISA (R&D Systems,Peprotech or Life Technologies) or Luminex multiplex kits (Procarta LifeTechnologies). The T-cells were stained for a panel of surface markersincluding checkpoint inhibitors (e.g., CTLA4) and evaluated by flowcytometry for Cbl-b inhibitor effects.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from exhausted T-cells (e.g., GM-CSF,IFNγ, and IL-2) in the presence of myeloid cells, which was indicativeof decreased T-cell exhaustion. Cbl-b inhibitors were also tested fortheir effects on checkpoint modulator expression levels followingactivation of exhausted T-cells.

Human T-Cell In Vitro Models of T-Cell Anergy

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary T-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-096-535 (i.e., cocktail of antibodies against surface markers CD14,CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a were incubatedwith the PBMCs before passing the samples by magnetic beads for removalof cells expressing those surface markers) or Stemcell TechnologiesCatalog #17951) to yield>95% CD3+ cells assessed by flow cytometry. Thecells were activated with immobilized anti-CD3 antibody (OKT3) andsoluble anti-CD28 antibody (28.2) for two days at which time they werewashed and allowed to rest for three days in the absence of stimulation.They were then treated with ionomycin (Sigma) for 18-24 hours to induceanergy. Following two washes to remove the ionomycin from the samples,Cbl-b inhibitor compounds were added to the cells at the indicatedconcentrations (e.g., 10, 1.11, and 0.37 μM) and incubated for 1 hour.The cells were then re-challenged with anti-CD3 antibody and anti-CD28antibody for 24 hours at which point cell free supernatants werecollected and assessed for cytokines (e.g., IFNγ) by ELISA (R&D Systemsor Peprotech) or Luminex multiplex kits (Procarta Life Technologies)following the manufacturer's protocols.

Cbl-b inhibitors were tested to determine their ability to induce orenhance secretion of cytokines from anergic T-cells (e.g., IFNγ), whichwas indicative of decreased T-cell tolerance.

In Vivo Activity of Cbl-b Inhibitors

A method of determining the pharmacodynamic profile of Cbl-b inhibitorswas performed by dosing strains of mice with competent immune systemssuch as C57BL/6 or BALB/c with a Cbl-b inhibitor. The Cbl-b inhibitorwas dissolved in a suitable formulation and administered by one ofvarious routes such as intravenous (IV), intraperitoneal (IP),subcutaneous (SC), or oral (PO), at a suitable dose level and frequency(e.g., twice per day BID or thrice per day TID) as informed by priorpharmacokinetic and tolerability studies. Following administration ofthe Cbl-b inhibitor, T-cells and indirectly other immune cells (e.g.,via cytokine production) were stimulated in vivo by administration of ananti-CD3 antibody or antigen-binding fragment thereof in PBS at definedamounts such as 2 μg or 10 μg per animal by routes such as IV or IP (seeHirsh et al., J. Immunol., 1989; Ferran, et al., Eur. J. Immunol.,1990). Additional study control arms included groups of mice treatedwith a vehicle formulation alone (i.e., formulation without the Cbl-binhibitor and anti-CD3 antibody), a formulation containing the Cbl-binhibitor alone, a formulation containing the anti-CD3 antibody alone,PBS alone, or combinations of these agents. The level of immuneactivation was then assessed by analysis of plasma cytokine levelsand/or expression of activation markers on immune cells (e.g., T-cells).Blood or lymphoid organs (e.g., spleen) were collected at defined timepoints (e.g., 8 hours or 24 hours). Blood samples were processed tocollect plasma for determination of cytokine levels using standardmethods known in the art. Cytokines measured included IL-2, IFNγ, andTNFα. Additional blood samples and lymphoid tissues were processed forflow cytometric analysis of immune cells (e.g., T-cells) using standardmethods to determine expression of cell type-specific markers andactivation markers such as CD25 and/or CD69. Augmentation of immunestimulation by Cbl-b inhibitor administration was assessed by comparingthe relative concentrations of cytokines in plasma, or the expressionlevels of activation markers on immune cells between appropriate groups(e.g., mice treated with Cbl-b inhibitor and 2 μg anti-CD3 antibodyversus mice treated with vehicle and 2 μg anti-CD3 antibody).

Cbl-b inhibitors were tested to determine their ability to induce orenhance the level of cytokines (e.g., IL-2, IFNγ, and TNFα) in bloodobtained from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response. Cbl-b inhibitorswere also tested to determine their ability to induce or enhance theexpression of cell surface markers on T-cells (e.g., CD25 and/or CD69)isolated from treated mice stimulated with an anti-CD3 antibody, whichwas indicative of modulation of the immune response.

B-Cell Activation Assay

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Human primary B-cells were isolatedfrom the PBMCs utilizing negative selection with commercial kitsfollowing the manufacturer's protocol (Stemcell Technologies Catalog#17954) to yield>95% CD20+ cells assessed by flow cytometry. Primaryhuman B-cells were plated at 0.7-1×10⁵ per well in a 96-well plate withCbl-b inhibitors over a dose ranging from 10 μM to 1 nM and incubated at37° C. 5% CO₂, with a final DMSO concentration of <0.5%. Cells werestimulated with anti-IgM for 20 hours at 37° C. 5% CO₂. Surfaceactivation markers on mature CD20⁺ IgD⁺ B-cells were monitored by FACSusing an anti-CD69 antibody (BD Biosciences).

Cbl-b inhibitors were tested to determine their ability to induce orenhance surface expression of cell surface markers on B-cells (e.g.,CD69), which was indicative of B-cell activation.

Purification and Activation of Primary Human NK-Cells.

Peripheral blood mononuclear cells (PBMC) were obtained either: 1) byusing Ficoll-Paque™ (GE Healthcare) for separation of peripheral bloodhematopoietic cells from buffy coats of healthy human donors; or 2)directly from LeukoPak donations. Total human primary NK-cells wereisolated from the PBMCs utilizing negative selection with commercialkits following the manufacturer's protocol (Miltenyi Biotec Catalog#130-092-657 or Stemcell Technologies Catalog #17955) to yield>92%CD56+, CD3-cells as assessed by flow cytometry. The cells were culturedovernight with IL-2 (60 ng/mL) at 37° C. 5% CO₂. Cbl-b inhibitors wereadded 1 hour prior to stimulation and incubated at 37° C. 5% CO₂ at aspecific concentration (e.g., 10 μM, 1 μM, or 0.1 μM) with a final DMSOconcentration of <0.1%. NK-cells were co-cultured with target cells thatwere engineered to have a red nucleus (K562 NucRed) measurable by flowcytometry. K562 NucRed cells were produced by transduction of K562 cellswith IncuCyte NucLight Red Lentivirus reagent (Catalog #4476) andselected for 5 days. Clonal populations were isolated and expanded usingstandard tissue culture techniques, and individual clones were validatedby comparison to wildtype K562 cells in NK-cell killing assays. Thecells were mixed at the indicated ratios (e.g., 5:1, 1:1, or 1:5) of NK(effector cells) to K562 NucRed (target cells) for 6 hours. Cell freesupernatants were collected and analyzed for cytokine secretion (e.g.,TNFα, IFNγ, or MIP1β) by ELISA or Luminex multiplex kits following themanufacturer's protocol. IFNγ secretion was assessed using an R&DSystems ELISA kit (Catalog #DY285), TNFα secretion was assessed using anR&D Systems ELISA kit (Catalog #DY210), and MIP1β secretion was assessedusing an R&D Systems ELISA kit (Catalog #DY271).

Biological Example 15: Evaluation of a Cbl-b Inhibitor in Combinationwith an Oncolytic Virus for Treating Cancer

Oncolytic viruses preferentially infect and kill cancer cells andstimulate host anti-tumor immune responses. This example describes theevaluation of a combination therapy including an oncolytic virus and aCbl-b inhibitor for treating cancer in mice.

Materials and Methods

In brief, the combination therapy was tested in C57BL/6 mice bearing asyngeneic MC-38 cell tumor. MC-38 cells were derived from a murineadenocarcinoma, which had been induced by subcutaneous injection ofdimethylhydrazine (Cameron et al., J Exp Med, 171: 249-263, 1990). MC-38cells were injected subcutaneously or intraperitoneally. Tumors wereallowed to grow for about 5-7 days at which time the animals wererandomized and treatment started.

An oncolytic virus, such as vaccinia virus, was administeredintraperitoneally at a dose of about 10⁸-10⁹ plague forming units(Puhlmann et al., Cancer Gene Therapy, 7: 66-73, 2000). The Cbl-binhibitor was dissolved in a suitable formulation and administered at asuitable dose level and frequency as informed by prior pharmacokineticand tolerability studies. In initial studies, a Cbl-b inhibitor in asuitable vehicle, or the vehicle alone, was administered orally (PO) ata dose of 180 mg/kg twice per day (BID) beginning on day 0 (=day 5-7post MC-38 injection). In further studies, a Cbl-b inhibitor formulationwas administered orally (PO) or parenterally (e.g., IV, IP, SC, orintratumorally at one to three injection sites per tumor). In additionto the test group of mice who received the combination therapy (theCbl-b inhibitor formulation plus the oncolytic virus), the studyincluded control groups of mice who received: the vehicle formulationplus DPBS, the Cbl-b inhibitor formulation plus DPBS, or the vehicleformulation plus the oncolytic virus.

The level of response was evaluated by measuring tumor growth andcomparing tumor growth in the test mice versus the control mice. Inaddition, the level of immune activation was assessed by collectingtumors for analysis of tumor infiltrating lymphocytes (TILs). In furtherexperiments, PBMCs or splenocytes were also collected. TILs andoptionally other lymphocyte samples were processed for flow cytometricanalysis using standard methods to determine antigen specificity,expression of cell type-specific markers, and expression of activationmarkers such as granzyme B, CD25, CD69, PD-1, and LAG3. Augmentation ofthe anti-tumor immune response by the combination therapy was assessedby comparing the relative percentage of immune cell populations in thetumor, and the relative levels of expression of activation markers onimmune cells in mice of the test and study groups.

Biological Example 16: Evaluation of Vaccine Material and Methods

Mice were implanted with 50,000 TC-1 cells/mouse subcutaneously into theright flank (at Day Zero (DO)). Thirteen days later (D13), when tumorsmeasured approximately 50-100 mm³, mice from appropriate groups (10 miceper group) were injected with HPV16 vaccine (s.c., total 2 doses, at D13and Day 20 (D20)). Compound 8 was reconstituted in 0.5% Methylcellulose,0.2% Polysorbate 80 and was administered orally from D13 to Day 35 (D35)at indicated doses. The group of mice that received the vaccine alsoreceived an oral vehicle (0.5% Methylcellulose, 0.2% Polysorbate 80).Mice were euthanized when tumor exceeded 1500-2000 mm³, according toIACUC guidelines. Survival was compared using GraphPad Prism usinglog-rank (Mantel-Cox) test. **=p<0.005 of treatment vs. Vehicle+Vaccinecontrol group at Day 61.

TC-1 cells were derived by stable transfection of mouse lung epithelialcells with human papillomavirus strain 16 (HPV16) early proteins 6 and 7(E6 and E7) and activated h-ras oncogene. Cells were obtained from Dr.T-C Wu (Johns Hopkins University). (Lin K Y, Guarnieri F G,Staveley-O'Carroll K F, Levitsky H I, August J T, Pardoll D M, et al.Treatment of established tumors with a novel vaccine that enhances majorhistocompatibility class II presentation of tumor antigen. Cancer Res1996; 56:21-6).

The HPV16 vaccine was prepared as follows: The CTL epitope from HPV16E749-57 (9 amino acid (aa) peptide, RAHYNIVTF, 100 μg/mouse, SEQ ID NO:2) was mixed with synthetic T helper epitope PADRE (13 aa peptide,aK-Cha-VAAWTLKAAa, SEQ ID NO: 3, where “a” is D alanine and Cha isL-cyclohexylalanine, 20 ug/mouse) and with QuilA adjuvant (20 ug/mouse).

Results

To evaluate the antitumor therapeutic response of Compound 8, the TC-1syngeneic mouse model was used. This model is poorly immunogenic andrequires vaccination to generate an effector CD8+T cell immune response(Cancer Immunol Res. 2017 September; 5(9):755-766). HPV16 vaccinationgenerates a CD8+T cell response against E7 tumor antigen. FIGS. A and Bshow the effects of the vaccine in combination with Compound 8 on tumorgrowth and survival. Although the vaccine alone minimally affected tumorgrowth (FIGS. A and B), Compound 8 in combination with vaccine led tosignificant slowdown of tumor progression (FIG. A) and was associatedwith prolonged survival (FIG. B). These results demonstrate thatCompound 8 significantly improved the E7-specific CD8+ T cell responseinduced by the vaccine, leading to an antitumor response and prolongedsurvival in tumor bearing mice.

The disclosures of all publications, patents, patent applications, andpublished patent applications referred to herein by an identifyingcitation are hereby incorporated herein by reference in their entirety.

Although aspects of the foregoing disclosure have been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainchanges and modifications will be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of thedisclosure.

What is claimed is:
 1. A compound of Formula (I):

or a tautomer thereof, stereoisomer thereof, or a pharmaceuticallyacceptable salt thereof, wherein

is

X is CH or nitrogen; Z¹ is CH or nitrogen; Z² is CH or nitrogen; R^(1a)and R^(1b) are independently hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, orC₁-C₆ alkyl-OH; R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkyl-OH, C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl); R^(2b)is hydrogen, halo, or C₁-C₆ alkyl; or R^(2a) and R^(2b) are takentogether with the carbon atom to which they are attached to form a spiro3- to 6-membered heterocyclyl or a spiro C₃-C₆ cycloalkyl, wherein atleast one of the atoms of the spiro heterocyclyl which is adjacent tothe connecting piperidinyl ring is carbon; R^(3a) and R^(3b) areindependently hydrogen, halo, or C₁-C₆ alkyl; or R^(3a) and R^(3b) aretaken together with the carbon atom to which they are attached to formC₃-C₄ cycloalkyl; R⁴ is hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆alkyl-OH;

is

and Y is CR^(5a)R^(5b) or sulfur; or

is

and Y is a bond; W is oxygen or a bond; R^(5a) and R^(5b) areindependently hydrogen, halo, or C₁-C₆ alkyl; or R^(5a) and R^(5b) aretaken together with the carbon atom to which they are attached to form aC₃-C₆ cycloalkyl; R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆cycloalkyl; R⁷ is hydrogen, halo, C₃-C₆ cycloalkyl, —NH-(3- to6-membered heterocyclyl), —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl),—O-(3- to 6-membered heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆cycloalkyl); R⁸ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl; R⁹is hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and R¹⁰ is—CF₃ or cyclopropyl.
 2. The compound of claim 1, wherein

is


3. The compound of claim 2, wherein Z¹ is CH.
 4. The compound of claim3, wherein

is


5. The compound of claim 2, wherein Z¹ is nitrogen.
 6. The compound ofclaim 5, wherein

is


7. The compound of claim 1, wherein

is


8. The compound of claim 7 wherein Z² is CH.
 9. The compound of claim 8,wherein

is


10. The compound of claim 7, wherein Z² is nitrogen.
 11. The compound ofclaim 10, wherein

is


12. The compound of any one of claims 1-11, wherein

is

and Y is CR^(5a)R^(5b) or sulfur.
 13. The compound of claim 12, whereinW is a bond.
 14. The compound of claim 12, wherein W is oxygen.
 15. Thecompound of any one of claims 1-11, wherein

is


16. The compound of claim 15, wherein R⁸ is C₁-C₃ alkyl, C₁-C₃haloalkyl, or C₃-C₄ cycloalkyl.
 17. The compound of claim 16, wherein R⁸is —CH₃, —CF₃, or cyclopropyl.
 18. The compound of any one of claims1-17, wherein X is CH.
 19. The compound of any one of claims 1-17,wherein X is nitrogen.
 20. The compound of any one of claims 1-19,wherein Y is a bond.
 21. The compound of any one of claims 1-19, whereinY is CR^(5a)R^(5b).
 22. The compound of claim 21, wherein R^(5a) andR^(5b) are independently hydrogen, halo, or C₁-C₃ alkyl, or R^(5a) andR^(5b) are taken together with the carbon atom to which they areattached to form a C₃-C₄ cycloalkyl.
 23. The compound of claim 22,wherein R^(5a) and R^(5b) are independently hydrogen, fluorine, or —CH₃;or R^(5a) and R^(5b) are taken together with the carbon atom to whichthey are attached to form cyclopropyl.
 24. The compound of claim 23,wherein R^(5a) and R^(5b) are independently hydrogen or fluorine. 25.The compound of any one of claims 1-19, wherein Y is sulfur.
 26. Thecompound of any one of claims 1-25, wherein R^(1a) and R^(1b) areindependently hydrogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃ alkyl-OH.27. The compound of claim 26, wherein R^(1a) and R^(1b) areindependently hydrogen, —CH₃, —CF₃, or —CH₂OH.
 28. The compound of anyone of claims 1-27, wherein R^(1b) is hydrogen.
 29. The compound of anyone of claims 1-28, wherein R^(2a) is —CN, C₁-C₃ alkyl, C₁-C₃ haloalkyl,C₁-C₃ alkyl-OH, C₁-C₃ alkyl-CN, or —(C₁-C₃ alkylene)-O—(C₁-C₃ alkyl).30. The compound of claim 29, wherein R^(2a) is —CN, —CH₃, —CF₃, —CH₂OH,—CH₂CN, or —CH₂—O—CH₃.
 31. The compound of any one of claims 1-30,wherein R^(2b) is hydrogen, halo, or C₁-C₃ alkyl.
 32. The compound ofclaim 31, wherein R^(2b) is hydrogen, fluorine, or —CH₃.
 33. Thecompound of any one of claims 1-28, wherein R^(2a) and R^(2b) are takentogether with the carbon atom to which they are attached to form a spiro4- to 5-membered heterocyclyl or a spiro C₃-C₄ cycloalkyl.
 34. Thecompound of claim 33, wherein R^(2a) and R^(2b) are taken together withthe carbon atom to which they are attached to form spiro cyclopropyl,


35. The compound of any one of claims 1-34, wherein R^(3a) and R^(3b)are independently hydrogen, halo, or C₁-C₃ alkyl; or R^(3a) and R^(3b)are taken together with the carbon atom to which they are attached toform C₃-C₄ cycloalkyl.
 36. The compound of claim 35, wherein R^(3a) andR^(3b) are independently hydrogen, fluorine, or —CH₃; or R^(3a) andR^(3b) are taken together with the carbon atom to which they areattached to form cyclopropyl.
 37. The compound of any one of claims1-36, wherein R⁴ is hydrogen, C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃alkyl-OH.
 38. The compound of claim 37, wherein R⁴ is hydrogen, —CH₃,—CF₃, or —CH₂OH.
 39. The compound of any one of claims 1-38, wherein R⁶is C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₃-C₄ cycloalkyl.
 40. The compoundof claim 39, wherein R⁶ is —CH₃, —CHF₂, or cyclopropyl.
 41. The compoundof any one of claims 1-40, wherein R⁷ is hydrogen, halo, C₃-C₄cycloalkyl, —NH(4- to 5-membered heterocyclyl), —NH(C₁-C₃ alkyl),—NH(C₃-C₅ cycloalkyl), —O(C₁-C₃ alkyl), —O(4- to 5-memberedheterocyclyl), or —O(C₃-C₅ cycloalkyl).
 42. The compound of claim 41,wherein R⁷ is hydrogen, chlorine, cyclopropyl, —NH(CH₂CH₃),—NH(cyclopropyl), —OCH₂CH₃, —O(cyclopropyl),


43. The compound of any one of claims 1-42, wherein R⁹ is hydrogen,C₁-C₃ alkyl, C₁-C₃ haloalkyl, or C₁-C₃ alkyl-OH.
 44. The compound ofclaim 43, wherein R⁹ is hydrogen, —CH₃, —CF₃, or —CH₂OH.
 45. Thecompound of claim 1, wherein the compound is a compound of Formula(I-a):

or a tautomer thereof, stereoisomer thereof, or a pharmaceuticallyacceptable salt thereof, wherein:

is

X is CH or nitrogen; Z¹ is CH or nitrogen; Z² is CH or nitrogen; R^(1a)and R^(1b) are independently hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, orC₁-C₆ alkyl-OH; R^(2a) is —CN, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkyl-OH, C₁-C₆ alkyl-CN, or —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl); R^(2b)is hydrogen, halo, or C₁-C₆ alkyl; or R^(2a) and R^(2b) are takentogether with the carbon atom to which they are attached to form a spiro3- to 6-membered heterocyclyl or a spiro C₃-C₆ cycloalkyl, wherein atleast one of the atoms of the spiro heterocyclyl which is adjacent tothe connecting piperidinyl ring is carbon; R^(3a) and R^(3b) areindependently hydrogen, halo, or C₁-C₆ alkyl; or R^(3a) and R^(3b) aretaken together with the carbon atom to which they are attached to formC₃-C₄ cycloalkyl; R⁴ is hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆alkyl-OH;

is

and Y is CR^(5a)R^(5b) or sulfur; or

is

and Y is a bond; W is oxygen or a bond; R^(5a) and R^(5b) areindependently hydrogen, halo, or C₁-C₆ alkyl; or R^(5a) and R^(5b) aretaken together with the carbon atom to which they are attached to form aC₃-C₆ cycloalkyl; R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆cycloalkyl; R⁷ is hydrogen, C₃-C₆ cycloalkyl, —NH-(3- to 6-memberedheterocyclyl), —NH—(C₁-C₆ alkyl), —NH—(C₃-C₆ cycloalkyl), —O-(3- to6-membered heterocyclyl), —O—(C₁-C₆ alkyl), or —O—(C₃-C₆ cycloalkyl); R⁸is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₃-C₆ cycloalkyl; R⁹ is hydrogen,C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkyl-OH; and R¹⁰ is —CF₃ orcyclopropyl.
 46. The compound of claim 1, selected from Compound Nos.1-45 or 47-52 in Table
 1. 47. The compound of claim 1, selected fromCompound Nos. 47-52 in Table
 1. 48. A pharmaceutical compositioncomprising the compound of any preceding claim, and a pharmaceuticallyacceptable excipient.
 49. A method of modulating activity of an immunecell, the method comprising contacting the immune cell with an effectiveamount of a Cbl-b inhibitor to modulate activity of the immune cell,wherein the Cbl-b inhibitor is a compound of any one of claims 1-47. 50.The method of claim 49, wherein the immune cell comprises a T-cell, aB-cell, or a natural killer (NK)-cell.
 51. The method of claim 49 orclaim 50, wherein the immune cell has been or is isolated from a bloodsample from a mammalian subject.
 52. The method of claim 49 or claim 50,wherein the immune cell is a tumor infiltrating lymphocyte (TIL) thathas been or is isolated from a tumor of a mammalian subject with cancer.53. The method of any one of claims 49-52, wherein the immune cellcomprises a T-cell, and wherein modulating activity of the T-cellcomprises one or more of increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and decreased T-celltolerance.
 54. The method of claim 53, wherein increased T-cellactivation comprises increased production of a cytokine.
 55. The methodof claim 54, wherein the cytokine comprises one or more selected fromthe group consisting of IL-2, IFN-γ, TNFα, and GM-CSF.
 56. The method ofclaim 53 or 54, wherein increased T-cell activation comprises increasedcell surface expression of one or more T-cell activation markers. 57.The method of claim 56, wherein the T-cell activation markers compriseone or more selected from the group consisting of CD25, CD69, and CTLA4.58. The method of any one of claims 53-57, wherein the T-cell has beenor is in contact with an anti-CD3 antibody alone or in combination withan anti-CD28 antibody.
 59. The method of any one of claims 53-57,further comprising culturing the immune cell with IL-2 alone or incombination with an anti-CD3 antibody and/or an anti-CD28 antibody. 60.The method of any one of claims 49-52, wherein the immune cell comprisesa NK-cell, and wherein modulating activity of an NK-cell comprisesincreased NK-cell activation.
 61. The method of claim 60, whereinincreased NK-cell activation comprises increased production of acytokine.
 62. The method of claim 61, wherein the cytokine comprises oneor more selected from the group consisting of IFN-γ, TNFα, and MIP1β.63. The method of any one of claims 49-62, wherein the immune cellcomprises a B-cell, and wherein modulating activity of a B-cellcomprises increased B-cell activation, optionally wherein increasedB-cell activation comprises increased expression of CD69.
 64. The methodof any one of claims 49-63, wherein the immune cell is a human immunecell.
 65. The method of any one of claims 49-64, wherein the immune cellcomprises a recombinant chimeric receptor.
 66. The method of claim 65,wherein the recombinant chimeric receptor is a chimeric antigenreceptor.
 67. A method of producing a modified immune cell, comprisingculturing a cell population containing an immune cell in the presence ofan effective amount of a Cbl-b inhibitor to modulate activity of theimmune cell, thereby producing the modified immune cell, wherein theCbl-b inhibitor is a compound of any one of claims 1-47.
 68. The methodof claim 67, further comprising culturing the immune cell with ananti-CD3 antibody alone or in combination with an anti-CD28 antibody.69. The method of claim 67, further comprising culturing of the immunecell with IL-2 alone or in combination with an anti-CD3 antibody and/oran anti-CD28 antibody.
 70. The method of any one of claims 67-69,further comprising recovering the modified immune cell.
 71. The methodof any one of claims 67-70, wherein the immune cell has been or isisolated from a blood sample from a mammalian subject, or the immunecell is a tumor infiltrating lymphocyte (TIL) that has been or isisolated from a tumor of a mammalian subject with cancer.
 72. The methodof any one of claims 67-70, wherein the immune cell is a cell selectedfrom the group consisting of: a hematopoietic cell, a multipotent stemcell, a myeloid progenitor cell, a lymphoid progenitor cell, a T-cell, aB-cell, and a NK-cell.
 73. The method of any one of claims 67-70,wherein the modified immune cell is a cell selected from the groupconsisting of: a hematopoietic cell, a multipotent stem cell, a myeloidprogenitor cell, a lymphoid progenitor cell, a T-cell, a B-cell, and aNK-cell.
 74. The method of any one of claims 67-73, wherein the immunecell is a tumor infiltrating lymphocyte (TIL).
 75. The method of any oneof claims 67-74, wherein the immune cell is a human immune cell.
 76. Themethod of any one of claims 67-75, wherein the immune cell or modifiedimmune cell comprises a recombinant chimeric receptor.
 77. The method ofclaim 76, wherein the recombinant chimeric receptor is a chimericantigen receptor.
 78. A modified immune cell produced by the method ofany one of claims 67-77.
 79. A modified immune cell comprising a Cbl-binhibitor, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47.
 80. An isolated modified immune cell, wherein the immunecell has been contacted or is in contact with a Cbl-b inhibitor, whereinthe Cbl-b inhibitor is a compound of any one of claims 1-47.
 81. Themodified immune cell of claim 79 or claim 80, wherein the immune cellhas been or is isolated from a blood sample from a mammalian subject, orthe immune cell is a tumor infiltrating lymphocyte (TIL) that has beenor is isolated from a tumor of a mammalian subject with cancer.
 82. Themodified immune cell of claim 80 or 81, wherein the immune cell is atumor-infiltrating lymphocyte (TIL) isolated from a tumor of a mammaliansubject with cancer before the immune cell is contacted with the Cbl-binhibitor.
 83. The modified immune cell of any one of claims 80-82,wherein the modified immune cell is a T-cell, and wherein the T-cellexhibits one or more of increased T-cell activation, increased T-cellproliferation, decreased T-cell exhaustion, and decreased T-celltolerance.
 84. The modified immune cell of claim 83, wherein increasedT-cell activation comprises increased production of a cytokine.
 85. Themodified immune cell of claim 84, wherein the cytokine comprises one ormore selected from the group consisting of IL-2, IFN-γ, TNFα, andGM-CSF.
 86. The modified immune cell of any one of claims 83-85, whereinincreased T-cell activation comprises increased cell surface expressionof one or more T-cell activation markers.
 87. The modified immune cellof claim 86, wherein the T-cell activation markers comprise one or moreselected from the group consisting of CD25, CD69, and CTLA4.
 88. Themodified immune cell of any one of claims 83-87, wherein the T-cell hasbeen or is in contact with an anti-CD3 antibody alone or in combinationwith an anti-CD28 antibody.
 89. The modified immune cell of any one ofclaims 83-87, wherein the T-cell has been or is in contact with IL-2alone or in combination with an anti-CD3 antibody and/or an anti-CD28antibody.
 90. The modified immune cell of any one of claims 80-82,wherein the modified immune cell is a NK-cell, and wherein the NK-cellexhibits increased NK-cell activation.
 91. The modified immune cell ofclaim 90, wherein increased NK-cell activation comprises increasedproduction of a cytokine.
 92. The modified immune cell of claim 91,wherein the cytokine comprises one or more selected from the groupconsisting of IFN-γ, TNFα, and MIP1β.
 93. The modified immune cell ofany one of claims 80-82, wherein the modified immune cell is a B-cell,and wherein the B-cell exhibits increased B-cell activation, optionallywherein increased B-cell activation comprises increased expression ofCD69.
 94. The modified immune cell of any one of claims 80-93, whereinthe modified immune cell is a human immune cell.
 95. The modified immunecell of any one of claims 80-94, wherein the modified immune cellcomprises a recombinant chimeric receptor.
 96. The modified immune cellof claim 95, wherein the recombinant chimeric receptor is a chimericantigen receptor.
 97. A composition comprising a cell populationcontaining the modified immune cell of any one of claims 78-96.
 98. Thecomposition of claim 97, further comprising a pharmaceuticallyacceptable excipient.
 99. The composition of claim 97, wherein thecomposition is in a culture vessel.
 100. The composition of claim 99,wherein the culture vessel is a tube, a dish, a bag, a multiwell plate,or a flask.
 101. The composition of claim 97 or 98, wherein thecomposition is in a suitable container.
 102. The composition of claim101, wherein the suitable container is a bottle, a vial, a syringe, anintravenous bag, or a tube.
 103. A method of modulating the immuneresponse, the method comprising administering an effective amount of themodified immune cell of any one of claims 78-96 or an effective amountof the composition of any one of claims 97-102 to an individual in needthereof.
 104. The method of claim 103, wherein the individual has acancer.
 105. A method of treating a cancer responsive to inhibition ofCbl-b activity, the method comprising administering an effective amountof the modified immune cell of any one of claims 78-96 or an effectiveamount of the composition of any one of claims 97-102 to an individualhaving the cancer responsive to inhibition of Cbl-b activity.
 106. Themethod of claim 104 or 105, wherein the cancer is a hematologic cancer.107. The method of claim 106, wherein the hematologic cancer is alymphoma, a leukemia, or a myeloma.
 108. The method of claim 104 or 105,wherein the cancer is a non-hematologic cancer.
 109. The method of claim108, wherein the non-hematologic cancer is a sarcoma, a carcinoma, or amelanoma.
 110. A method of inhibiting abnormal cell proliferation, themethod comprising administering an effective amount of the modifiedimmune cell of any one of claims 78-96 or an effective amount of thecomposition of any one of claims 97-102 to an individual in needthereof.
 111. The method of claim 110, wherein the abnormal cellproliferation is hyperplasia or cancer cell proliferation.
 112. Themethod of claim 111, wherein the cancer cell is from a hematologiccancer.
 113. The method of claim 112, wherein the hematologic cancer isa lymphoma, a leukemia, or a myeloma.
 114. The method of claim 111,wherein the cancer cell is from a non-hematologic cancer.
 115. Themethod of claim 114, wherein the non-hematologic cancer is a sarcoma, acarcinoma, or a melanoma.
 116. A method of modulating the immuneresponse, the method comprising administering an effective amount of aCbl-b inhibitor to an individual to modulate the immune response in theindividual, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47.
 117. A method of inhibiting Cbl-b activity, the methodcomprising administering an effective amount of a Cbl-b inhibitor to anindividual to inhibit Cbl-b activity in the individual, wherein theCbl-b inhibitor is a compound of any one of claims 1-47.
 118. A methodof treating a cancer responsive to inhibition of Cbl-b activity, themethod comprising administering an effective amount of a Cbl-b inhibitorto an individual to treat the cancer responsive to inhibition of Cbl-bactivity, wherein the Cbl-b inhibitor is a compound of any one of claims1-47.
 119. The method of claim 118, wherein the cancer is a hematologiccancer, optionally wherein the hematologic cancer is a lymphoma, aleukemia, or a myeloma.
 120. The method of claim 118, wherein the canceris a non-hematologic cancer, optionally wherein the non-hematologiccancer is a sarcoma, a carcinoma, or a melanoma.
 121. The method of anyone of claims 116-120, wherein the Cbl-b inhibitor is administered byenteral administration, optionally wherein the enteral administration isoral administration.
 122. The method of any one of claims 116-120,wherein the Cbl-b inhibitor is administered by parenteraladministration, optionally wherein the parenteral administration isintratumoral administration.
 123. The method of any one of claims118-122, further comprising administering an effective amount of themodified immune cell of any one of claims 78-96 or an effective amountof the composition of any one of claims 97-102 to the individual totreat the cancer.
 124. A method of inhibiting abnormal cellproliferation, the method comprising administering an effective amountof a Cbl-b inhibitor to an individual to inhibit abnormal cellproliferation in the individual, wherein the Cbl-b inhibitor is acompound of any one of claims 1-47.
 125. The method of claim 124,wherein the abnormal cell proliferation is hyperplasia or cancer cellproliferation.
 126. The method of claim 125, wherein the cancer cell isfrom a hematologic cancer, optionally wherein the hematologic cancer isa lymphoma, a leukemia, or a myeloma.
 127. The method of claim 125,wherein the cancer cell is from a non-hematologic cancer, optionallywherein the non-hematologic cancer is a sarcoma, a carcinoma, or amelanoma.
 128. The method of any one of claims 124-127, wherein theCbl-b inhibitor is administered by enteral administration, optionallywherein the enteral administration is oral administration.
 129. Themethod of any one of claims 124-127, wherein the Cbl-b inhibitor isadministered by parenteral administration, optionally wherein theparenteral administration is intratumoral injection, or the parenteraladministration is by a route selected from the group consisting ofintravenous, intraperitoneal, and subcutaneous.
 130. The method of anyone of claims 116-129, wherein the individual has one or more ofincreased T-cell activation, increased T-cell proliferation, decreasedT-cell exhaustion, and decreased T-cell tolerance after administrationof the Cbl-b inhibitor.
 131. The method of claim 130, wherein increasedT-cell activation comprises increased production of a cytokine.
 132. Themethod of claim 131, wherein the cytokine comprises one or more selectedfrom the group consisting of IL-2, IFN-γ, TNFα, and GM-CSF.
 133. Themethod of any one of claims 130-132, wherein increased T-cell activationcomprises increased cell surface expression of one or more T-cellactivation markers.
 134. The method of claim 133, wherein the T-cellactivation markers comprise one or more selected from the groupconsisting of CD25, CD69, and CTLA4.
 135. The method of any one ofclaims 116-134, wherein the individual has increased NK-cell activationafter administration of the Cbl-b inhibitor.
 136. The method of claim135, wherein increased NK-cell activation comprises increased productionof a cytokine.
 137. The method of claim 136, wherein the cytokinecomprises one or more selected from the group consisting of IFN-γ, TNFα,and MIP1β.
 138. The method of any one of claims 116-137, wherein theindividual has increased B-cell activation after administration of theCbl-b inhibitor, optionally wherein increased B-cell activationcomprises increased expression of CD69.
 139. A cell culture compositioncomprising a cell population containing an immune cell and a Cbl-binhibitor, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47.
 140. The cell culture composition of claim 139, wherein theimmune cell is a cell selected from the group consisting of: ahematopoietic cell, a multipotent stem cell, a myeloid progenitor cell,a lymphoid progenitor cell, a T-cell, a B-cell, and a NK-cell.
 141. Thecell culture composition of claim 139 or 140, further comprising ananti-CD3 antibody alone or in combination with an anti-CD28 antibody.142. The cell culture composition of any one of claims 139-141, whereinthe immune cell is an engineered immune cell comprising a recombinantchimeric receptor.
 143. The cell culture composition of claim 142,wherein the recombinant chimeric receptor is a chimeric antigenreceptor.
 144. A pharmaceutical composition comprising a Cbl-b inhibitorand one or both of an adjuvant and an antigen, wherein the Cbl-binhibitor is a compound of any one of claims 1-47.
 145. Thepharmaceutical composition of claim 144, wherein the antigen is a cancerantigen.
 146. An article of manufacture comprising the modified immunecell of any one of claims 78-96, the composition of any one of claims97-102, the cell culture composition of any one of claims 139-143, orthe pharmaceutical composition of claim
 48. 147. The article ofmanufacture of claim 146, wherein the modified immune cell or cellculture composition is in a tube, a dish, a bag, a multiwell plate, or aflask.
 148. The article of manufacture of claim 146, wherein themodified immune cell or pharmaceutical composition is in a bottle, avial, a syringe, an intravenous bag, or a tube.
 149. A kit comprisingthe modified immune cell of any one of claims 78-96 or the compositionof any one of claims 97-102.
 150. The kit of claim 149, wherein themodified immune cell is in a tube, a dish, a bag, a multiwell plate, ora flask.
 151. The kit of claim 149, wherein the modified immune cell isin a bottle, a vial, a syringe, an intravenous bag, or a tube.
 152. Thekit of any one of claim 149-151, wherein the kit comprises instructionsfor administering the modified immune cell or composition to anindividual according to the method of any one of claim 103-115.
 153. Akit comprising the pharmaceutical composition of claim
 48. 154. The kitof claim 153, wherein the kit comprises instructions for administeringthe pharmaceutical composition to an individual according to the methodof any one of claims 116-118.
 155. A kit comprising the cell culturecomposition of any one of claims 139-143.
 156. The kit of claim 155,wherein the kit comprises instructions for producing a modified immunecell according to the method of any one of claims 67-77.
 157. A methodfor treating or preventing a disease or condition associated with Cbl-bactivity, the method comprising administering a Cbl-b inhibitor to anindividual in need thereof, wherein the Cbl-b inhibitor is a compound ofany one of claims 1-47.
 158. Use of a Cbl-b inhibitor in the manufactureof a medicament for treating or preventing a disease or conditionassociated with Cbl-b activity, wherein the Cbl-b inhibitor is acompound of any one of claims 1-47.
 159. Use of a Cbl-b inhibitor in themanufacture of a medicament for treating cancer, wherein the Cbl-binhibitor is a compound of any one of claims 1-47.
 160. A Cbl-binhibitor for use in treating or preventing a disease or conditionassociated with Cbl-b activity, wherein the Cbl-b inhibitor is acompound of any one of claims 1-47.
 161. A Cbl-b inhibitor for use intreating cancer, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47.
 162. A method of treating cancer, the method comprising:administering to an individual with cancer an effective amount of aCbl-b inhibitor, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47, and administering to the individual an effective amount ofan additional therapeutic agent.
 163. The method of claim 162, whereinthe Cbl-b inhibitor and the additional therapeutic agent areadministered consecutively in either order.
 164. The method of claim162, wherein the Cbl-b inhibitor and the additional therapeutic agentare administered concurrently.
 165. The method of any one of claims162-164, wherein the additional therapeutic agent comprises an immunecheckpoint inhibitor.
 166. The method of claim 165, wherein the immunecheckpoint inhibitor is an antagonist of at least one inhibitorycheckpoint molecule selected from the group consisting of PD-1 (CD279),PD-L1 (CD274), CTLA-4 (CD125), LAG3 (CD223), PVR (CD155), PVRL2 (CD112),PVRL3 (CD113), TIGIT, TIM3 (CD366), and VISTA.
 167. The method of claim165, wherein the immune checkpoint inhibitor is an antagonist of atleast one inhibitory checkpoint molecule selected from the groupconsisting of PD-1 (CD279), PD-L1 (CD274), and CTLA-4 (CD152).
 168. Themethod of claim 167, wherein the at least one inhibitory checkpointmolecule comprises PD-1, optionally wherein the immune checkpointinhibitor is selected from the group consisting of pembrolizumab,nivolumab, cemiplimab, and biosimilars thereof.
 169. The method of claim167, wherein the at least one inhibitory checkpoint molecule comprisesPD-L1, optionally wherein the immune checkpoint inhibitor is selectedfrom the group consisting of atezolizumab, avelumab, durvalumab, andbiosimilars thereof.
 170. The method of claim 167, wherein the at leastone inhibitory checkpoint molecule comprises CTLA-4, optionally whereinthe immune checkpoint inhibitor is selected from the group consisting ofipilimumab, tremelimumab, and biosimilars thereof.
 171. The method ofany one of claims 165-167, wherein the immune checkpoint inhibitorcomprises an antibody or antigen-binding fragment thereof, optionallywherein the antibody or fragment is human or humanized.
 172. The methodof any one of claims 162-171, wherein the additional therapeutic agentcomprises an antineoplastic agent.
 173. The method of claim 172, whereinthe antineoplastic agent is classified as one of the group consisting ofa cytotoxic antibiotic, a plant alkaloid, an antimetabolite, analkylating agent, and other antineoplastic agent.
 174. The method ofclaim 173, wherein the antineoplastic agent comprises a cytotoxicantibiotic, optionally wherein the cytotoxic antibiotic is selected fromthe group consisting of ixabepilone, mitomycin, plicamycin, bleomycin,pixantrone, amrubicin, valrubicin, pirarubicin, mitoxantrone,idarubicin, zorubicin, aclarubicin, epirubicin, daunorubicin,doxorubicin, and dactinomycin.
 175. The method of claim 173, wherein theantineoplastic agent comprises a plant alkaloid, optionally wherein theplant alkaloid is selected from the group consisting of trabectedin,cabazitaxel, paclitaxel poliglumex, docetaxel, paclitaxel, demecolcine,teniposide, etoposide, vintafolide, vinflunine, vinorelbine, vindesine,vincristine, and vinblastine.
 176. The method of claim 173, wherein theantineoplastic agent comprises an antimetabolite, optionally wherein theantimetabolite is a pyrimidine analog, a purine analog, or a folic acidanalog, optionally wherein the antimetabolite is selected from the groupconsisting of floxuridine, trifluridine, tegafur, fluorouracil,decitabine, azacitidine, capecitabine, gemcitabine, carmofur, tegafur,fluorouracil, cytarabine, nelarabine, clofarabine, fludarabine,cladribine, tioguanine, mercaptopurine, pralatrexate, pemetrexed,raltitrexed, and methotrexate.
 177. The method of claim 173, wherein theantineoplastic agent comprises an alkylating agent, optionally whereinthe alkylating agent is selected from the group consisting ofdacarbazine, temozolomide, pipobroman, mitobronitol, etoglucid, uracilmustard, ranimustine, nimustine, fotemustine, streptozocin, semustine,lomustine, carmustine, carboquone, triaziquone, thiotepa, mannosulfan,treosulfan, busulfan, bendamustine, prednimustine, trofosfamide,ifosfamide, mechlorethamine, melphalan, chlorambucil, andcyclophosphamide.
 178. The method of claim 173, wherein theantineoplastic agent comprises an other antineoplastic agent selectedfrom the group consisting of a platinum compound, a protein kinaseinhibitor, and an other agent.
 179. The method of claim 178, wherein theantineoplastic agent comprises a platinum compound, optionally whereinthe platinum compound is selected from the group consisting ofcisplatin, carboplatin, oxaliplatin, satraplatin, and polyplatillen.180. The method of claim 178, wherein the antineoplastic agent comprisesa protein kinase inhibitor.
 181. The method of claim 178, wherein theantineoplastic agent comprises an other agent.
 182. The method of anyone of claims 162-181, further comprising administering to theindividual an effective amount of radiation therapy.
 183. A method oftreating cancer, the method comprising: administering to an individualwith cancer an effective amount of a Cbl-b inhibitor, wherein the Cbl-binhibitor is a compound of any one of claims 1-47, and administering tothe individual an effective amount of radiation therapy.
 184. The methodof claim 182 or claim 183, wherein the radiation therapy is externalbeam radiation therapy.
 185. The method of claim 182 or claim 183,wherein the radiation therapy is internal radiation therapy.
 186. Themethod of any one of claims 162-185, wherein the Cbl-b inhibitor isadministered by enteral administration, optionally wherein the enteraladministration is oral administration.
 187. The method of any one ofclaims 162-185, wherein the Cbl-b inhibitor is administered byparenteral administration, optionally wherein the parenteraladministration is intratumoral injection, or the parenteraladministration is by a route selected from the group consisting ofintravenous, intraperitoneal, and subcutaneous.
 188. The method of anyone of claims 162-187, wherein the cancer is a hematologic cancer. 189.The method of claim 188, wherein the hematologic cancer is a lymphoma, aleukemia, or a myeloma.
 190. The method of any one of claims 162-187,wherein the cancer is a non-hematologic cancer.
 191. The method of claim190, wherein the non-hematologic cancer is a carcinoma, a sarcoma, or amelanoma.
 192. A method of producing an expanded population of tumorinfiltrating lymphocytes (TILs), the method comprising: (a) obtaining abiological sample comprising TILs from the individual treated accordingto the method of any one of claims 162-191, and (b) culturing the TILsin cell culture medium comprising at least one T-cell growth factor toproduce an expanded population of TILs.
 193. The method of claim 192,wherein the at least one T-cell growth factor comprises IL-2.
 194. Themethod of claim 192 or claim 193, wherein the cell culture mediumfurther comprises an anti-CD3 antibody, or both an anti-CD3 antibody andan anti-CD28 antibody.
 195. The method of any one of claims 192-194,wherein the cell culture medium further comprises the Cbl-b inhibitor.196. The method of any one of claims 192-195, wherein the cell culturemedium further comprises irradiated feeder cells.
 197. The method of anyone of claims 162-196, wherein the individual is a human patient.
 198. Acomposition comprising the expanded population of TILs produced by themethod of any one of claims 192-197, and a physiologically acceptablebuffer.
 199. A method of treating cancer, the method comprisingadministering an effective amount of the composition of claim 198 to theindividual with cancer.
 200. The method of claim 199, further comprisingcontinuing to administer an effective amount of the Cbl-b inhibitor tothe individual.
 201. A method of producing an expanded population oftumor infiltrating lymphocytes (TILs), the method comprising: (a)obtaining a biological sample comprising TILs from an individual havingcancer who has received or is receiving an effective amount of a Cbl-binhibitor, wherein the Cbl-b inhibitor is a compound of any one ofclaims 1-47; and (b) culturing the TILs in cell culture mediumcomprising at least one T-cell growth factor to produce an expandedpopulation of TILs.
 202. The method of claim 201, wherein the at leastone T-cell growth factor comprises IL-2.
 203. The method of claim 201 orclaim 202, wherein the cell culture medium further comprises an anti-CD3antibody, or both an anti-CD3 antibody and an anti-CD28 antibody. 204.The method of any one of claims 201-203, wherein the cell culture mediumfurther comprises the Cbl-b inhibitor.
 205. The method of any one ofclaims 201-204, wherein the cancer is a non-hematologic cancer,optionally wherein the non-hematologic cancer is a sarcoma, a carcinoma,or a melanoma.
 206. The method of any one of claims 201-205, wherein theindividual is a human patient.
 207. A composition comprising theexpanded population of TILs produced by the method of any one of claims201-206, and a physiologically acceptable buffer.
 208. A method oftreating cancer, the method comprising administering an effective amountof the composition of claim 207 to the individual with cancer.
 209. Themethod of claim 208, further comprising continuing to administer aneffective amount of the Cbl-b inhibitor.
 210. A method of immunizing,the method comprising: administering to an individual in need thereof aneffective amount of a small molecule Cbl-b inhibitor, and administeringto the individual an effective amount of a vaccine.
 211. The method ofclaim 210, wherein the immunizing is a method of treating cancer,comprising: administering to an individual with cancer an effectiveamount of a small molecule Cbl-b inhibitor, and administering to theindividual an effective amount of a therapeutic cancer vaccine.
 212. Themethod of claim 211, wherein the Cbl-b inhibitor and the cancer vaccineare administered consecutively.
 213. The method of claim 211, whereinthe Cbl-b inhibitor and the cancer vaccine are administeredconcurrently.
 214. The method of any one of claims 211-213, wherein thecancer vaccine is an immunogenic composition comprising at least onetumor antigen and a pharmaceutically acceptable excipient.
 215. Themethod of claim 214, wherein the at least one tumor antigen comprises atleast one synthetic peptide or recombinant protein.
 216. The method ofclaim 214 or claim 215, wherein the immunogenic composition furthercomprises an adjuvant.
 217. The method of claim 216, wherein theadjuvant comprises one or more ingredients of the group consisting ofaluminum salts, squalene, and saponins.
 218. The method of claim 214 orclaim 215, wherein the immunogenic composition further comprisesantigen-presenting cells (APCs), optionally wherein the APCs aredendritic cells, optionally wherein the cancer vaccine is PROVENGE. 219.The method of any one of claims 211-214, wherein the cancer vaccinecomprises a microbial vector, optionally wherein the microbial vector isTICE-BCG.
 220. The method of claim 219, wherein the microbial vector isa recombinant viral vector or a recombinant bacterial vector.
 221. Themethod of any one of claims 211-214, wherein the cancer vaccinecomprises killed cancer cells or a cancer cell lysate.
 222. A method oftreating cancer, the method comprising: administering to an individualwith cancer an effective amount of a small molecule Cbl-b inhibitor, andadministering to the individual an effective amount of an oncolyticvirus.
 223. The method of claim 222, wherein the Cbl-b inhibitor and theoncolytic virus are administered consecutively.
 224. The method of claim222, wherein the Cbl-b inhibitor and the oncolytic virus areadministered concurrently.
 225. The method of any one of claims 222-224,wherein the oncolytic virus is a virus selected from the groupconsisting of adenovirus, coxsackievirus, echovirus, fowlpox virus,herpes simplex virus, maraba virus, measles virus, myxoma virus,Newcastle disease virus, parvovirus, poliovirus, retrovirus, reovirus,Seneca Valley virus, Semiliki Forest virus, vaccinia virus, andvesicular stomatitis virus.
 226. The method of any one of claims222-225, wherein the oncolytic virus is a recombinant virus comprisingone or both of a functional deletion of at least one viral gene, and aninsertion of at least one transgene.
 227. The method of claim 226,wherein the recombinant virus comprises a functional deletion of atleast one viral gene and an insertion of at least one transgene. 228.The method of claim 226 or claim 227, wherein the transgene encodeshuman granulocyte macrophage colony-stimulating factor (GM-CSF). 229.The method of any one of claims 222-226, wherein the oncolytic virus isa transgenic serotype 5 adenovirus.
 230. The method of any one of claims222-228, wherein the oncolytic virus is a transgenic herpes simplexvirus type-1 (HSV-1), optionally wherein the transgenic HSV-1 istalimogene laherparepvec.
 231. The method of any one of claims 222-228,wherein the oncolytic virus is a transgenic vaccinia virus, optionallywherein the transgenic vaccinia virus is pexastimogene devacirepvec.232. The method of any one of claims 222-226, wherein the oncolyticvirus is a non-recombinant oncolytic virus, optionally wherein thenon-recombinant oncolytic virus is a virus selected from the groupconsisting of an echovirus, a Newcastle disease virus, a parvovirus, areovirus, and a Seneca Valley virus.
 233. The method of any 211-232,wherein the cancer is a hematologic cancer.
 234. The method of claim233, wherein the hematologic cancer is a lymphoma, a leukemia, or amyeloma.
 235. The method of 234, wherein the cancer is a non-hematologiccancer.
 236. The method of claim 235, wherein the non-hematologic canceris a carcinoma, a sarcoma, or a melanoma.
 237. The method of any210-236, wherein the small molecule Cbl-b inhibitor is a compound of anyone of claims 1-47.
 238. The method of any 210-237, wherein the smallmolecule Cbl-b inhibitor is a compound selected from the compounds inTable 1 and/or Table 1A, or a tautomer thereof, stereoisomer thereof, ora pharmaceutically acceptable salt thereof.
 239. A method of treatingcancer, the method comprising: administering to an individual withcancer an effective amount of an agent capable of lowering activationthreshold of an immune cell, and administering to the individual aneffective amount of a therapeutic cancer vaccine; or administering tothe individual an effective amount of an oncolytic virus.
 240. Themethod of claim 239, wherein the agent is further capable of reducingcostimulation requirement of an immune cell.
 241. The method of claim239 or claim 240, wherein the agent is further capable of promotingtumor immune-surveillance.
 242. The method of any one of claims 239-241,wherein the agent is a small molecule, Cbl-b inhibitor.
 243. Apharmaceutical composition comprising a cancer vaccine and a smallmolecule Cbl-b inhibitor, optionally wherein the composition furthercomprises a pharmaceutically acceptable excipient.
 244. A kit fortreating cancer, the kit comprising: (a) a small molecule Cbl-binhibitor; (b) a therapeutic cancer vaccine; (c) instructions foradministration of an effective amount of the Cbl-b inhibitor and thetherapeutic cancer vaccine to treat cancer in an individual.
 245. A kitfor treating cancer, the kit comprising: (a) a pharmaceuticalcomposition comprising a small molecule Cbl-b inhibitor and atherapeutic cancer vaccine; and (b) instructions for administration ofan effective amount of the pharmaceutical composition comprising theCbl-b inhibitor and the therapeutic cancer vaccine to treat cancer in anindividual.
 246. The method, composition, or kit of any one of claims239-245, wherein the cancer vaccine is an immunogenic compositioncomprising at least one tumor antigen and a pharmaceutically acceptableexcipient.
 247. The method, composition, or kit of claim 246, whereinthe at least one tumor antigen comprises at least one synthetic peptideor recombinant protein.
 248. The method, composition, or kit of claim246 or claim 247, wherein the immunogenic composition further comprisesan adjuvant.
 249. The method, composition, or kit of claim 248, whereinthe adjuvant comprises one or more ingredients of the group consistingof aluminum salts, squalene, and saponins.
 250. The method, composition,or kit of claim 246 or claim 247 wherein the immunogenic compositionfurther comprises antigen-presenting cells (APCs), optionally whereinthe APCs are dendritic cells, optionally wherein the cancer vaccine isPROVENGE.
 251. The method, composition, or kit of claim 246, wherein thecancer vaccine comprises a microbial vector, optionally wherein themicrobial vector is TICE-BCG.
 252. The method, composition, or kit ofclaim 251, wherein the microbial vector is a recombinant viral vector ora recombinant bacterial vector.
 253. The method, composition, or kit ofany one of claims 239-246, wherein the cancer vaccine comprises killedcancer cells or a cancer cell lysate.
 254. A pharmaceutical compositioncomprising an oncolytic virus and a small molecule Cbl-b inhibitor,optionally wherein the composition further comprises a pharmaceuticallyacceptable excipient.
 255. A kit for treating cancer, the kitcomprising: (a) a small molecule Cbl-b inhibitor; (b) an oncolyticvirus; (c) instructions for administration of an effective amount of theCbl-b inhibitor and the oncolytic virus to treat cancer in anindividual.
 256. A kit for treating cancer, the kit comprising: (a) apharmaceutical composition comprising a small molecule Cbl-b inhibitorand an oncolytic virus; and (b) instructions for administration of aneffective amount of the pharmaceutical composition comprising the smallmolecule Cbl-b inhibitor and the oncolytic virus to treat cancer in anindividual.
 257. The method, composition, or kit of any one of claims239-242, or 254-256, wherein the oncolytic virus is a virus selectedfrom the group consisting of adenovirus, coxsackievirus, echovirus,fowlpox virus, herpes simplex virus, maraba virus, measles virus, myxomavirus, Newcastle disease virus, parvovirus, poliovirus, retrovirus,reovirus, Seneca Valley virus, Semiliki Forest virus, vaccinia virus,and vesicular stomatitis virus.
 258. The method, composition, or kit ofany one of claims 239-242, or 254-256, wherein the oncolytic virus is arecombinant virus comprising one or both of a functional deletion of atleast one viral gene, and an insertion of at least one transgene. 259.The method, composition, or kit of claim 258, wherein the recombinantvirus comprises a functional deletion of at least one viral gene and aninsertion of at least one transgene.
 260. The method, composition, orkit of claim 258 or claim 259, wherein the transgene encodes humangranulocyte macrophage colony-stimulating factor (GM-CSF).
 261. Themethod, composition, or kit of any one of claims 239-242 or 254-258,wherein the oncolytic virus is a transgenic serotype 5 adenovirus. 262.The method, composition, or kit of any one of claims 239-242 or 254-260,wherein the oncolytic virus is a transgenic herpes simplex virus type-1(HSV-1), optionally wherein the transgenic HSV-1 is talimogenelaherparepvec.
 263. The method, composition, or kit of any one of claims239-242 or 254-260, wherein the oncolytic virus is a transgenic vacciniavirus, optionally wherein the transgenic vaccinia virus is pexastimogenedevacirepvec.
 264. The method, composition, or kit of any one of claims239-242 or 254-260, wherein the oncolytic virus is a non-recombinantoncolytic virus, optionally wherein the non-recombinant oncolytic virusis a virus selected from the group consisting of an echovirus, aNewcastle disease virus, a parvovirus, a reovirus, and a Seneca Valleyvirus.
 265. The method, composition, or kit of any one of claims239-264, wherein the cancer is a hematologic cancer.
 266. The method,composition, or kit of claim 265, wherein the hematologic cancer is alymphoma, a leukemia, or a myeloma.
 267. The method, composition, or kitof any one of claims 239-264, wherein the cancer is a non-hematologiccancer.
 268. The method, composition, or kit of claim 267, wherein thenon-hematologic cancer is a carcinoma, a sarcoma, or a melanoma. 269.The method, composition or kit of any one of claims 239-268, wherein thesmall molecule Cbl-b inhibitor is a compound of any one of claims 1-47.270. The method, composition, or kit of any one of claims 239-269,wherein the small molecule Cbl-b inhibitor is a compound selected fromthe compounds in Table 1 and/or Table 1A, or a tautomer thereof,stereoisomer thereof, or a pharmaceutically acceptable salt thereof.